Single-Stranded Binding Proteins
Table Of Contents
- DNA Replication
- DNA Repair and Recombination
- Single-Stranded Binding Proteins
- Crystal Structure
- Clinical Effects of Malfunction
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DNA Replication
DNA Replication is one of the three main processes which Single-Stranded Binding Proteins are involved with. This is the process in which the complementary strands comprising the double helix are separated and serve as templates for new copies of DNA. Replication is catalyzed by an enzyme called DNA Polymerase, which requires a 3'-OH primer and moves in a 5' to 3' direction adding new complementary nucleotides (A, T, G, and C) to the 3' end of the growing strand. Replication occurs at the replication fork, and both template strands are processed in this area. Helicases are the hexamer proteins which use ATP hydrolysis to separate the double-stranded DNA, making the single-stranded DNA available for DNA polymerase to replicate. Both strands are processed in a 5' to 3' direction, with the leading strand being replicated continuously and the lagging strand having discontinuous replication (processed in chunks called Okazaki fragments which are sealed together by DNA Ligase). The difference in processing of the leading and lagging strands is necessary due to the antiparallel nature of the double helix (Watson et al., 2008).
There are three general steps to note during DNA replication:
1. Unwinding of parent DNA (accomplished by Helicases)
2. Formation of short primer sequence complementary to template strands (RNA Polymerase involved here)
3. Elongation of primer sequence by DNA Polymerase, forming a new daughter strand (Lodish et al., 2008)
Video overview of DNA Replication
DNA Repair and Recombination
SSBs are also thought to play a role in methyl-directed mismatch repair, interacting with Helicase, Exouncleases, and Polymerase. An overview of mismatch repair depicts the roles of these enzymes. Mismatch repair is the process by which incorrectly paired bases are cleaved from damaged DNA. SSBs are also involved in DNA recombination, where SSB is thought to help facilitate RecA binding to ssDNA. Recombination is the process by which new DNA is formed by the incorporation of DNA from homologous chromosomes. The first step in DNA recombination involves the RecBCD complex separating the dsDNA until it reaches a chi site. At this point, the strand is cleaved and strand invasion occurs, leading to recombinant DNA. Finally, strand resolution ends the recombination process, reforming dsDNA (mmbr.asm.org).
Single-Stranded Binding Protein
Single Stranded Binding Proteins (SSBs) are an important factor in DNA Replication. SSBs follow behind the Helicase and bind to single-stranded DNA, stabilizing the strands so that DNA Polymerase can bind effectively to replicate the DNA. SSBs serve to prevent the ssDNA from re-annealing before DNA Polymerase replicates (Watson et al., 2008). SSBs also exhibit cooperative binding, where the binding of one SSB promotes the binding of another. Studies involving E. coli have shown that SSBs have a higher binding affinity to polynucleotides which already have SSB bound, indicating E. coli SSB cooperativity. The binding interaction between SSBs on the ssDNA strand leads to greater strength of stabilization for the ssDNA and ensures that ssDNA is coated with SSBs rapidly after being separated by Helicase (mmbr.asm.org).
There is a relatively basic chemical mechanism for binding of SSB to ssDNA, which involves electrostatic interactions with the DNA phosphate backbone, and stacking interactions with individual bases. What separates SSBs from other DNA binding proteins is the fact that it does not make many hydrogen bonds with these bases (Watson et al., 2008).
Figure (shown above) depicting the state of single stranded DNA before and after SSB binding during replication
Research has been conducted to determine the effect of SSBs on the accuracy of in vitro DNA synthesis in Escherichia coli, and results showed that the presence of SSBs increased the accuracy of DNA replication, resulting in up to a 10 fold increase in effectiveness of replication. This increase was found to be progressive, that is the accuracy and results of synthesis improved with the concentration of SSB utilized in replication. This leads to the conclusion that the increase in rigidity of the DNA strand based on interaction between the strand and the SSB may significantly increase accuracy of replication. (www.pnas.org)
Figure depicting the replication fork as a whole (including Helicase, SSBs, Primase, and DNA Polymerase positions on unwound DNA strand)
Crystal Structure
The crystal structure of a protein depicts the detailed components of the protein, including secondary structural components such as alpha helices and beta pleated sheets. Although crystal structure images depict similar aspects of proteins, there is not one uniform crystal structure for single stranded binding proteins in general as these proteins vary in structure based on the organism and cellular components they are associated with.
The crystal structure (shown above) of SSB in Thermotoga maritima, a rod-shaped marine sedimentary bacterium (interscience.wiley.com)
View the crystal structure of SSB of Human RepA protein (topsan.org)
Clinical Effects of Malfunction
Research has been conducted to determine the effects of the absence of SSBs in Escherichia coli bacteria. Results showed that a lack of SSB led to a greater incidence of deletions within CTG triplet repeats. Increased SSB leads to decreased formation of secondary DNA structures. Individuals with triplet repeat diseases show instability in CGG and CTG repeats (much higher numbers of these sequences), and this may be due to an increased likelihood of secondary structure formation within these triplet repeats (jb.asm.org).
Testing showed that properly functional SSB was necessary in maintaining stability of CGG and CTG triplet repeats in Escherichia coli. Specifically, the formation of hairpin structures (a form of secondary structure), which leads to increased mutagenesis and genetic error, was much higher in the test group utilizing the mutated form of SSB (jb.asm.org).
Many inheritable genetic diseases have been associated with an overexpression of CTG or CCG triplet repeats within a gene. For example, Myotonic Dystrophy (the most common adult-onset form of muscular dystrophy) is associated with the CNBP gene, which is repeated about 26 times in normal individuals and expressed overabundantly, anywhere from 75 to 11,000 times (nlm.nih.gov). Fragile X Syndrome, also known as Fragile X Mental Retardation, is another inheritable human disease related to extensive trinucleotide repeats, having 55 to 200 CGG repeats rather than the normal number of about 30 CGG repeats (www.fragilex.org). A third human disease related to triplet repeats is Huntington's Disease, a degenerative neurological disease. Again with Huntington's Disease, expansion of CAG repeats within the HTT gene lead to the disorder (www.wikipedia.org)
References
Lodish, Harvey et al. Molecular Cell Biology. 6th ed. New York: W.H. Freeman & Co., 2008. 139-145.
Watson, James D. et al. Molecular Biology of the Gene. 6th ed. San Francisco: Pearson Education, Inc.,2008. 195-220.
Comments (1)
Christopher Korey said
at 10:09 pm on Apr 6, 2009
Looks good. As you convert the outline to text, Remember to be concise about each subsection and provide link outs to other pages or papers that provide more in depth detail if that is required. If someone wants more information give them a way to find it not necessarily put it on the page. Try to divide the sections by inserting a horizontal bar. Remember to reference just like any other paper. Make the references links rather than pasting the URL into the reference list.
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