This is an interactive review sheet that encourages you to interact with the material and draw out the critical information.  There are several ways to contribute:

 

• Add an important vocabulary word and its definition to the list from a specific lecture.  These are anything you feel is essential to the understanding of the material including the names and functions of proteins, types of chemical bonds, names of processes, etc.

• Add an answer to one of the concept questions

• Edit and expand on another student's posting

• Add a link to another website, a video on You Tube, or any other teaching material on the web that you think enhances your understanding of the material.

 

As I stated in class, 8 points of your exam grade is dependent on your participation in this dialog about the material.  I will use the following rubric for grading: 3 points for adding and defining one vocabulary word, 3 points for participating in the answering of one of the concept questions, 2 points for editing and expanding on another student's posting (edits must be more than just removing a word or adding a word, it must have an impact on the material discussed)

 

Contributions to this review must be completed by 5 p.m. on March 10th (the Tuesday before the exam)

 

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Lecture 6 DNA Replication II: Origins and Telomeres

 

Important Vocabulary

 

Origins of Replication- The site where replication is initiated. This is not a random occuring event and the site occurs at the same positions on the DNA molecule. In prokaryotic, the OriC contains two short repeat motifs:

9mer- a 9 nucleotide repeat sequence. 5 copies are dispersed throughout the origin of replication and function as the binding site for the DnaA protein.

13-mer- A-T rich 13 nucleotide sequence occuring as three copies in tandem along the oriC where the double helix is actually melted

 

Telomerase - A 2 part enzyme that adds Tandem repeats (TTAGGGTTAGGG... in humans) to the telomere region of the chromosome on both the lagging ( Okazaki  fragment strand) and leading strand. Identified by Blackburn and Greider.  Telomerase assures shortening of the DNA does not occure everytime DNA replicates.  The overexpression of telomerase is linked with the development of many cancers.

 

     Part 1 - made up of protein. Has catalytic activity with no primer.

     Part 2 - "TERT" - RNA template that is species specific

 

Replicon- A segment of chromosomal DNA that consists of the origin of replication site (DNA A binds here to initiate replication) and all of the DNA that it controls the replication of. In bacteria the entire chromosome is typically one replicon since they usually have only one origin of replication site. In eukaryotes many replicons make up the entire chromosome.

 

Telomere- Stretches of G-rich DNA containing no genes and acting as protection for the ends of DNA. Telomeres have species specifc tandem repeats. In humans, the tandem repeat is 5' TTAGGG 3' . They are important because without them there would be deletions of the genetic code at the ends of DNA during replication.

 

DNA A: an initiator, associated with prokaryotic initiation, which uses ATP hydrolysis to facilitate the opening of DNA at a specific binding region. It can only function when it is bound to ATP

 

Prepriming complex DNA BC- DNA B is the helicase, while DNA C is the helicase loader that plays a transitory role  and is thus released from complex. Once this complex adds on the helicase, the initiation phase is completed.

 

PreRC:  Pre-replication complex; it is composed of the ORC, Cdc6, Cdt1, and two Mcm2-7 proteins.  Once the preRC is in place the DNA is licensed.This initiates firing of DNA replication after G1 phase.

 

Recombination Sites: specific short sequences where DNA exchange occurs; the two corresponding recombination sites have exactly the same nucleotide sequences

 

Methylation: occurs after prokaryotic initiation; the process of adding a methyl group onto the Adnines of all GATC sequences found in DNA during replication.  DAM Methylase catalyzes this reaction.  Old DNA is methylated, while new DNA is not, thus the double helix during replication is hemimethylated.  The new strand will be methylated after about ten minutes, but this lag is important in mismatch repair in order to distinguish between old and new strands.

 

CDK: cyclin dependent kinase- uses kinases and ATP to trigger protein destruction in order for the origin to fire. Also helps to control the the cell cycle depending on the concentration of CDK. CDk concentrations low during G1 and high during the rest of the cell cycle.

 

Important Concepts

 

How did they figure out there were 4 DNA polymerases at one origin with 2 forks?

The chromosome in E. Coli  was uniformly radioactively labeled with H3-thymidine.  After inspection, the chromosome showed one single origin of with two forks.  However, to determine directionality of replication, the procedure had to be altered.  During the replication process, the specific activity of the label in the growth medium was increased, which resulted in the most active regions of DNA synthesis being more heavily labeled.  After inspection, both replication forks were labeled heavily, which indicated that the replication was bidirectional.  If only one of the replication forks was heavily labeled, then it would have meant that unidirectional replication was occurring. 

 

How are prokaryotic and eukaryotic initiation similar?

Both prokaryotic and eukaryotic initiation utilize proteins which bind to a location on the DNA strand and facilitate helix opening. In prokaryotes, this is DNA A and in Eukaryotes, this is the ORC (origin recognition complex)/preRC (pre-replication complex). Both use multiple proteins for initiation, prokaryotic needing DnaA,B,C which are the initiator protein, the helicase, and the helicase loader; eukaryotics multiple initiation proteins are ORC, Cdc6 and Cdt1, which similarily are the initiator protein, the helicase and the helicase loader.

 

How are they different?

There is one replicon in bacteria, while Eukaryotes have multiple replicons. Prokaryotes utilize DNA A as an initiator, which binds to 9mers (DNA binding sequence) and utilizes ATP hydrolysis to facilitate opening of DNA. Around 30 DnaA molecules associate with the origin. This occurs at a specific binding region. Eukaryotic initiation utilizes the ORC (origin recognition complex) which is similar to DNA A in prokaryotes. The ORC facilitates opening of DNA and recruits other proteins to form a pre-replication complex. Eukaryotic initiation is also tied to the cell cycle, and firing of the origin of replication is based on the cell's location in the cell cycle. Also in Eukaryotes, different regions of DNA replicate at different times in the cycle, each region utilizing the ORC.

 

How are origins of replication prevented from firing more than once in Prokaryotes?

Seq A prevents the binding of DNA A again because it binds to the hemi-methylated groups on the new DNA.  When Seq A is removed, after additional time, DAM methylase binds. DnaA can bind only when both Seq A and DAM methylase are not present, which initiates replication again.  To start the process of intiation once more DnaA again requires the transformation of ADP + Pi, attached to the DnaA, back to ATP, which is a slow process.

 

 

What is the system that prokaryotes use that prevents DNA-A from re-binding to the origin of replication until time has passed? 

 

Prokaryotes Replication is prevented from firing more than once by the binding of SeqA to Hemi-Methlylated DNA ( Hemi-Methlylated = 1 old strand methlylated + 1 new strand NOT methlylated).  SeqA is replaced by Dam methylase, which methlylates the new strand of DNA. DnaA cannot bind to the initiation site when SeqA or Dam Methylase are present on the DNA. After both SeqA and Dam Methylase have fallen off, only then can DnaA rebind to the initiation site to start the replication process again.

 

 

How are origins of replication prevented from firing more than once in Eukaryotes?

Eukaryotes prevent origins of replication from firing more than once by utilizing Cyclin Dependent Kinase (CDK). CDK adds a phosphate to a protein involved in the cell cycle and is found at high levels in the S, G2, and M phases of the cell cycle, and at low levels in the G1 phase of the cell cycle. When CDK adds a phosphate to the Cdc6 protein it causes the protein to be broken into pieces.

 

 

How does telomerase solve the chromosome end problem in Eukaryotes?

Telomerase extends the 3' end of the telomere so that the additional 3' end can act as a template for a new Okazaki fragment on the 5' end.  Telomerase does this by matching its RNA template with the ssDNA of the telomere.  The sequence of the RNA template is repeating, so there is an unbound portion of the template beyond the 3' end of the ssDNA.  It is at this unbound portion that nucleotides bind, extending the 3' end of the ssDNA.  Upon release of the telomerase, the ssDNA of the telomere is paired with a new fragment on the 5' end of the telomere.  The Okazaki fragment is then repaired leaving the telomere with an extension (still having a 3' overhang).

 

 

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Lecture 7 DNA Repair

 

Important Vocabulary

 

Mismatch Repair- One self checking method in DNA replication.  In mismatch repair, MutS clamps on to the DNA phosphate backbone and runs down the strand.  It identifies the distortion that occurs in the helix when two bases are incorrectly paired.  MutS then forms a complex with MutL which stabilizes the DNA and together they recruit MutH which binds to the hemimethylated DNA and nicks the new strand on either side of the mistake so that it can be cut back and filled in properly.

 

Base Excision Repair- DNA repair system that uses DNA glycosylases (8 enzymes for common damage in humans) that recognize different damaged bases and breaks their glycosidic bonds revealing an APurinc Site (AP site) from the base reflecting outward which then DNA polymerase replaces the base at the 3'OH end repairing the damage.  This is followed by ligation by DNA ligase.

 

Nucleotide excision repair- DNA repair mechanism similar to base excision repair except it doesn't involve the removal of a damaged base. It involves more broader specificity and can deal with more damaged areas of the DNA.

 

UVR - Complex of proteins used in prokaryotic Nucleotide Excision Repair:

          A - identifies the lesion

          B - uses ATP hydrolysis to "melt" helix open

          C - recognizes & cuts DNA

          D - acts like helicase - unwinds forked DNA structures

 

Photoreactivation - The process whereby dimerized pyrimidines  in DNA are restored by an enzyme (deoxyribodipyrimidine photolyase) that requires light energy.

 

DNA photolyase - The enzyme used in photoreactiviation which uses visible light energy (at wavelengths between 300-500nm) to bind to cyclobutyl dimers. Once the enzyme binds it then converts the dimers back to their original monomeric nucleotides

 

DNA glycosylase- Enzyme important to base excision repair.  DNA glycosylase removes the nitrogenous bases from damaged nucleotides by flipping the base out of the DNA helix and chemically removing the purine or pyrimidine base.  Only the sugar-phosphate backbone is left at what is known as an AP-site.

 

Non-homologous End Joining- This is the repair process of double stranded DNA breaks. The name indicates that the ends of the molecules joining together do not need to be the identical. This is also looked at as a type of recombination because it can be used to join fragments that weren’t previously joined, thus producing new combinations.

 

MutS - A protein that exists as a dimer when it binds to the phosphate backbone of DNA during mismatch repair.  This protein runs along a strand of DNA and scans for base pairs that are mismatched (such as A-C or G-T). Base pairs that are mismatched create a distortion in the double helix of DNA and this distortion is recognized by the MutS protein. When the MutS protein encounters a mismatched base pair, it recruits MutL and MutH proteins and repairs the nucleotide. 

 

Important Concepts

 

What are the common themes for all the different repair mechanisms?

 

All of the different repair mechanisms operate with different proteins and processes. However, the general idea of how the mistake is corrected remains the same. In general, the damaged DNA is recognized due to the misalignment of hydrogen bond donors and receptors, which forms a bulge. The proteins are then able to distinguish between the undamaged strand and damaged strand. The damaged DNA is then cut out in a way that allows DNA polymerase to add the correct sequence of nucleotides. DNA ligase then seals up the backbone and the repair is complete.

 

How is the lesion differentiated from the correct DNA sequence in Mismatch Repair?

E. coli has three mismatch repair systems: long patch, short patch, and very short patch. In long patch systems the

MutS can detect a distortion, but the repair mechanism must be able to tell which strand includes the correct sequence.  MutL has the ability to find the strand that is hemimethylated.  This strand is the original template as the new strand takes some time to become hemimethylated.  This information is translated to MutH which nicks the incorrect strand. During short and very short mismatch repair, a similar situation is occurring. The difference here are the proteins that recognize the mismatch. The short patch system uses MutY to recognize an A-G or A-C mismatch and very short repair system corrects G-T mistakes. G-T mistakes are recognized by Vsr endonuclease.

 

 

How is the lesion differentiated from the correct DNA sequence in Base Excision Repair?

DNA Glycosylases, 8 enzymes important for detecting/cleaving common damage, identifies the lesion and cleaves the β-N-glycosidic bond between the damaged base and the sugar component of the nucleotide. The DNA Glycosylase removes a damaged base by ‘flipping' the structure to a position outside of the helix and then detaching it from the polynucleotide, this creates an AP site. These AP sites are recognised by AP endonuclease enzymes that complete the repair. DNA polymerase comes in and uses base-pairing with the undamaged base in the other strand of the helix to make sure that the right nucleotide is inserted.

 

How is the lesion differentiated from the correct DNA sequence in Nucleotide Excision Repair?

The UVR proteins present during Nucleotide Excision Repair allow for the recognition and repair of a distortion within the DNA sequence. UVR A identifies the lesion present within the DNA. UVR B then melts the helix open and allows the UVR C proteins to cleave the distored DNA (Each UVR C protein makes an excision on its respective side- being either on the 5' or 3' side of the detected lesion. The excision made on the 5' side is made about 8 nucleotides away from the distortion while the excision made on the 3' side is made about 4 nucleotides away). UVR D (also referred to as DNA helicase II) then helps to excise the now cleaved distored DNA. After which, DNA polymerase and DNA ligase come in to fill in and seal the gap.

 

What are the health consequences of inherited mutations in repair pathway proteins?

When mutations occur in repair pathway proteins, they result in severe health consequences.  When defects occur in nucleotide-excision repair, this can result in Xeroderma pigmentosum (sensitivity to sunlight=increased chance of skin cancer), Cockayne syndrome (Dwarfism, premature aging, mental retardation,etc...), and Trichothiodystrophy (skin abnormalities, brittle hair, short stature,etc...).  When defects occur in mismatch repair, it can lead to colon cancer.  When defects occur in repair of interstrand cross-links, it can result in Fanconi anemia (increased skin pigmentation, predisposition to leukemia.  Also damage may occur to DNA damage detection and response, which can lead to Ataxia telangiectasia (defective muscular coordination, immune deficiencies, = increased chance of cancer) and Li-Fraumeni syndrome (predisposition to cancer in different tissues).  When DNA repair Helicases are defective, it can result in Bloom syndrome (sun sensitivity, dwarfism, lead to cancer), Werner syndrome (premature aging and reduced lifespan), and Rothmund-Thomson Syndrome (sun sensitivity, short stature, and leads to cancer).

 

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Lecture 8 General Recombination

 

Important Vocabulary

 

General Recombination- A mechanism that exchanges DNA between a pair of homologous DNA sequences.  It is also referred to as homologous recombination. The general recombination reaction is essential for every proliferating cell, because accidents occur during nearly every round of DNA replication that interrupt the replication fork and require general recombination mechanisms to repair.

 

Strand Invasion- A required process of general recombination where the single stranded DNA is placed between the strands of the double stranded DNA. This invasion creates a "triple strand". After this invasion, the rec A protein coats the invaded ssDNA and allows the single strand to pair with its homologous strand within the dsDNA.

 

Rec BCD- This is a complex of proteins responsible for generating recombinogenic ends in bacteria. RecB and RecD are helicases that move down ssDNA (B moves 3' to 5' while D moves 5' to 3'). They also act as exonucleases, cutting the DNA as they move down the strand. RecC recognizes the Chi Site. Bacteria contain of 1000 of these sites which function to increase recombination.

 

Rec A- protein that is required to perform strand invasion.  It coats the single stranded DNA and allows pairing of homologous sequences.  It moves fastest from 5'-3' and slowest from 3'-5'. Does not recognize specific sequences, but recognizes specific conformations such as holiday junctions.

 

Heteroduplex DNA- incorrect pairing of 2 ssDNA strands from homologs which is fixed by mismatch repair. A heteroduplex is a double-stranded molecule of nucleic acid, such as DNA, which has arised through the genetic recombination of single complementary strands which are from different sources.  These sources could be different homologous sequences in one organism, or even sequences from different organisms.  Any problems are fixed by mismatch repair.

 

Migration and Resolution Proteins:

     Ruv A- binds junction (recognizes it)

     Ruv B- similar to helicase, uses ATP to move junction

     Ruv C- an endonuclease that cuts 2 strands, resolving the junction

   *This is better explained by this link: http://en.wikipedia.org/wiki/RuvABC*

 

Transduction- the process by which genetic  material (wherebeit bacterial, viral, or both DNA) is inserted into a cell by a viral agent as a bacteriophage. The viral vector provides a stable introduction or injection of a foreign gene into a host cell's genome.

 

Branch Migration- The process in which a crossover point between two DNA duplexes slides along the duplexes;  Moves the holliday junction back and forth so there is an increase or decrease in heteroduplex DNA.  Run by RUV A and RUV B proteins.

 

Vertical cut-  in resolution of general recombination, recombination results in "spliced" ends.  When the "Splice" or crossover products results in the reassortment of flanking genes.  Both vertical and horizontal cuts are equally likely.  This cut is made by the protein RuvC.

 

Horizontal cut-  In resolution of general recombination, recombination results in "patched" or crossover products with no reassortment.  Both vertical and horizontal cuts are equally likely.  This cut is made by the protein RuvC.

 

Gene Conversion:

An event in DNA genetic recombination usually occurring during meiosis, during mismatch repair with two different chromosomes. DNA sequence information is transferred from one DNA helix (which remains unchanged) to another DNA helix, whose sequence is altered. One chromatid is altered to be identical to the other causing unexpected outcomes (ex: 3:1 instead of 2:2).

 

Spo11- A protein used during meiotic recombination; involved with cutting DNA to start recombination.  Similar to Topoisomerase I in that it forms a phosphotyrosine bond between the protein and DNA.

 

Recombinogenic ends- after the endonuclease cuts both strands of the double helix, and the 5' ends at the break are chewed back by the exonuclease, the 3' end protrudes and will search for a homologous DNA helix with which to pair, beginning recombination process.

 

Holliday Junction-this is also known as a cross-stand exchange.  The holliday junction has a pair of strands that are crossing and another pair of strands that are noncrossing.  The holliday junction allows for strands to interact in order for recombination to occur.

 

Important Concepts

 

How are the repair of double strand breaks (DSBs) and production of DSBs for recombination connected?

     During recombination in meiosis, the two homologous DNA have a "cross-over."  The double strands break and then bind to the opposite strand to form a new double helix, each containing parts of the first two DNA molecules.  The repair of DSBs and the production of DSBs are similar because they both involve breaking chromosome ends that expose a single strand with the 3' end overhanging.  Also in both cases, the overhanging single-strand looks for a region of unbroken DNA with the same base sequence. 

 

How does the general model of strand invasion, branch migration, and resolution of the Holiday junction work?

Sister Cromatids first pair to make chromosomes, then two homologous chromosomes pair at the Metaphase plate. In order to facilitate recombination a double stranded break must occur in one of the homologous pairs. RedBCD recognizes the double stranded break site and acts as an endonuclease to "chew away" the strands. RecBCD continues to degrade the stands until RecC recognizes a Chi site which terminates degradation providing a free 3' end on the bottom strand. RacA facilitates strand invasion, creating a triple stranded helix until the invading strand can bind to the DNA sequence of the opposing homologous pair. When the invading strand finds this sequence, the helix to which it is invading zippers the invading strand to one strand of the helix and the remaining strand is pushed out where it will bind to the origional DSB. This cross over forms a Holiday Junction. 

RuvA recognizes the shape of the Holiday Junction and binds to it. Two RuvB proteins use ATP to spin the DNA past eachother to facilitate branch migration. RuvC is an endonuclease that cuts the junction creating either vertical or horizontal resolution. 

 

Endonuclease activity of RecBCD is greater in the the 3'-5' strand, rather than the 5'-3' strand.

Invasion

 

Strand invasion is required in order for recombination to occur. After recombinogenic ends of the DNA have been generated the RecA protein facilitates strand invasion by forming a helix around ssDNA from the 5' to 3' end. RecA looks for a homologus sequence of DNA on the other chromosome while it binds to the ssDNA. When a match is found RecA will "push" the old strand down and allow the recombinating strand to replace it and bind with the homologus DNA. Therefore, a holiday junction is created where the two homologus strands cross. 

 

Branch Migration

 

Is the ability of the Holiday Junction to shift, thus allowing more or less Heteroduplex DNA in the recombination process.  This translocation of the Holliday Junction along DNA progressively exchanges one DNA strand for another and determines the amount of information that is transferred between two recombining strands.  RUV –AB,&C proteins are responsible for the migration and resolution of this recombination process.

 

Resolution

In order to get two separate double helices during general recombination, the holiday junction must be resolved. Depending on whether the holiday junction is cut horizontally (the ends stay the same and the middle is what has been recombined) or cut vertically, the products can be either a "splice" (crossover products--> reassortment of flanking genes) or "patch" (noncrossover products-->no reassortment).

 

How do RecA and RecBCD function to promote recombination?

RecBCD binds to the free blunt ends of double stranded DNA, and moves along the dsDNA, unwinding it.  It acts as an endonuclease, degrading both single strands, until it encounters the chi site, where it's endonuclease activity changes.  It then creates a 3' whisker, where RecA aligns the ssDNA with it's homologous target region on the duplex DNA.  RecA then catalyzes the exchange of the incoming single strand for the corresponding strand of the duplex.

 

How is recombination initiated during meiosis in eukaryotes? Recombination is initated by the protein Spo11, an endonuclease.  It cuts the DNA during meiosis. Spo11 makes a double stranded cut in DNA to start recombination during meiosis. It is like Topo 1 in the sense that it makes a DNA protein double bond. A covalent bond is made and tyrosine is fused to start the break.  An exonuclease chews back the 5' ends of the DNA at the break leaving the 3' ends exposed, single-stranded, and protruding.  These protruding single-stranded 3' ends search for homologous base pair sequences in the double stranded portions of the other DNA.  When it finds a homologous base pair sequence, strand invasion occurs and recombination begins.

 

 

 

 

 

What is the connection between BRCA2, genome stability and cancer?  BRCA2 is in a class of genes known as tumor suppressor genes.  The BRCA2 protien recognizes the ends of chromosomes and recruits Rad51 to sites of damage and promotes repair. The damage it helps to control is associated with normal environmental factors, radiation, etc. Without the protien, there is an increased chance of having abnormal fusions which leads to inappropraite chromosomes.  This genomic instability can lead to uncontrolled cell division and the formation of tumors.  A lack of or mutation in the BRCA2 gene is often associated with an high risk of breast cancer.

 

 

 

 

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Lecture 9 Site-specific Recombination

 

Important Vocabulary

Recognition site- the specific site, in site specific recombination, in which recombination occurs. 

 

CRE recombinase- CRE pops out the chromosome from between lock sites that have the same orientation, bringing recognition sites together, the sequences pop out to form a circle; Cre recombinase(Cre) is a type 1 topoisomerase frm bacteriophage P1 that helps with site specific recombination of DNA.  Cre recombinase(Cre) is used in conjunction to modify chromosomes and genes.

 

Site Specific Recombination- recombination that occurs only at specific recognition sites, and uses recombinases to catalyze the exchange of DNA (doesn't use RecA). It also has a specific directionality.

 

Gene Knockout- The active use of recombination to eliminate part of a gene so that it will not make a protein. An organism is methodically reinforced so that it will carry genes that have been made inoperative or "knocked out" of the organism, such that there are no active genes present.

 

"Knock-in"-  Recombination is used to replace a gene with a mutant form of the gene so that only the mutant gene is active and the mutant protein is produced.

 

Integrases- are a diverse family of tyrosine recombinases which rearrange DNA duplexes by means of conservative site-specific recombination reactions; catalyzes Holliday junctions.

 

Insertion- When there is a circular phage; usually has one arrow (arrows show directionalitiy). And it is to be inserted into a linear DNA segment. The phage will insert with in the arrow on the linear DNA segment. Making one linear recombinant DNA chromosome.

 

Deleltion- There is a linear DNA segment, curves around and attaches at the arrows (where the lox sites are located.) What was on the outside of the arrows is now excised.

 

Inversion - When the linear DNA segment has arrows pointing opposite directions. The DNA segment that is inside the arrows will flip.

 

 

Important Concepts 

How do Tyrosine Recombinases Work?

After chromosomes are injected into bacteria, phage chromosomes become circular.  The attachment P (POP) or phage region aligns with the attachment B (BOB) or bacteria region.  Protein Int  then cuts(nicks) both chromosome strands at position "O" (5' - AAATATG- 3'). DNA sequence is tested before exchange in a triple-helical structure.  The strands then exchange and creates a Holliday junction.  Protein Int. mediates cleavage and strands are sealed.  The "O" region is about 7 basepairs long.  Integration results in the reorganizing of the order of genes on the phage chromosome.

 

How do Serine Recombinases work?

the serine recombinase ( a tetramer) binds to the DNA, forms a doublestranded break on both pieces of DNA, rotates so that the two helices are realigned, then seals the DNA to form two "new" molecules of DNA.  During this type of recombination, all four strands are cut through the recognition site.

 

What are the biological roles of Site Specific Recombination?  Site-specific recombination is used for bacterial genome replication, for integration of a bacteriophage into a host chromosome, for insertion of mobile genetic elements, or transposons, into a DNA sequence, to turn genes on and off, and to reversibly rearrange DNA.

What is needed in order for Site Specific Recombination to occur?

A recombination site (small DNA sequence), the recombinase recognition sequences, recombinase (which is a protein that catalyzes recombination), and a crossover region that is usually 7-20 basepairs long, are all required in order for site-specific recombination to occur.

 

How does site specific recombination differ from general recombination?

General recombination typically involves the cleavage and rejoining at identical or very similar sequences.  In site-specific recognition, cleavage will take place at a specific site into which DNA is inserted.  General recombination occurs in viruses during infection, as well as in bacteria during conjugation.  Site-specific recognition is involved in the parasitic distribution of DNA segments throughout ones genome.  Viruses rely on site-specific recombination to multiply and spread.  

General recombination is often referred to as homologous recombination.  This type of recombination is widely used in cells to repair double-strand breaks in DNA.  General recombination also produces new combinations of DNA sequences during crossover in meiosis.  These types of crossovers and exchange produce new combinations that produce genetic variation in populations as they reproduce. This allows evolutionary adaptation to different environments over time.

Site-specific recombination, which is also known as conservative site-specific recombination is a type of genetic recombination in which DNA strand exchange takes place between segments with little homology.  Recombinases rearrange the DNA segments by recognized and binding to short DNA sequences, where they cleave the DNA backbone and exchange the two DNA helices involved and then rejoin the strands.  This type of recombination is highly specific, fast, and efficient.  This type of recombination is used in molecular biology in bacterial genome replication, differentiation and movement of mobile genetic elements.  They are a potential basis for the development of genetic engineering tools.

 

What is Rett Syndrome?

Rett Syndrome is a neurodevelopmental disorder which is caused by mutations in the gene MECP2 found on the X chromosome.  It was first described by Andreas Rett of Austria in 1966.  Studies have shown that 95% of the mutations leading to this disorder orignate from mutated sperm.  This disorder mainly affects females(1:10,000), but if males have the disorder, which is rare, they often don't make it to term.  Since MECP2 is not required for early brain development but essential for maturing brain cells, infants with the disorder can often go 6-18 months with out detection and symptoms seem to progress with age.  Characteristics of Rett Syndrome include things such as screaming fits, lack of interest, loss of speech functions, balance/coordination problems, short stature, grinding of teeth, and hand stereotypies.  Currently there is no cure for Rett Syndrome, however some treatments include: social medications, SSRIs, anti- psychotics, speech and physical therapies.  Although males rarely survive to term, studies have shown females with Rett Syndrome can live up to 40 years or more.

 

What is a knock-out mouse?

A knock-out mouse is a mouse with a gene or genes removed at birth and then studied to see the effects from losing the particular gene or genes.  A knock-out mouse shows the function of the gene by what is impaired in the mouse. Knock-out mice are created by using homologous recombination to introduce an homologous sequence to the existing mouse DNA.  It is possible to create a knock-out mouse where a gene is not being expressed in a certain part of the body in a mouse, while in the rest of the mouse the gene is functioning normal, this is called a conditional knockout.  An example of a conditional knockout mouse whould be one that has been treated where the gene in not being expressed only in the brain but the gene in the rest of the body functions normly.  Scientist use conditional knockout when studying Rett mice.In order to create a conditional knockout mouse scientists use site specific recombination.