Replication protein A
{{Infobox heteropolypeptide
| heteropolymer = Replication protein A
| polymer_type = heterotrimer
| protein_type =
| function = damaged DNA binding, single-stranded DNA binding
| cofactors =
| image = 1L1O Replication protein A.png
| image_source = This is an image of human Replication protein A. From {{PDB|1L1O}} {{Proteopedia|Replication protein A}}
| SubunitCount = 3
| subunit1 = Replication protein A1
| gene1 = RPA1
| locus1 = Chr. 17 [https://www.ncbi.nlm.nih.gov/Omim/getmap.cgi?chromosome=17p13.3 p13.3]
| subunit2 = Replication protein A2
| gene2 = RPA2
| locus2 = Chr. 1 [https://www.ncbi.nlm.nih.gov/Omim/getmap.cgi?chromosome=1p35.3 p35.3]
| subunit3 = Replication protein A3
| gene3 = RPA3
| locus3 = Chr. 7 [https://www.ncbi.nlm.nih.gov/Omim/getmap.cgi?chromosome=7p21.3 p21.3]
}}
File:Steps in DNA synthesis.svg
Replication protein A (RPA) is the major protein that binds to single-stranded DNA (ssDNA) in eukaryotic cells.{{cite journal | vauthors = Wold MS | title = Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism | journal = Annual Review of Biochemistry | volume = 66 | issue = 1 | pages = 61–92 | year = 1997 | pmid = 9242902 | doi = 10.1146/annurev.biochem.66.1.61 }}{{cite journal | vauthors = Chen R, Wold MS | title = Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair | journal = BioEssays | volume = 36 | issue = 12 | pages = 1156–1161 | date = December 2014 | pmid = 25171654 | pmc = 4629251 | doi = 10.1002/bies.201400107 }} In vitro, RPA shows a much higher affinity for ssDNA than RNA or double-stranded DNA.{{cite journal | vauthors = Flynn RL, Zou L | title = Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 45 | issue = 4 | pages = 266–275 | date = August 2010 | pmid = 20515430 | pmc = 2906097 | doi = 10.3109/10409238.2010.488216 }} RPA is required in replication, recombination and repair processes such as nucleotide excision repair and homologous recombination.{{cite journal | vauthors = Caldwell CC, Spies M | title = Dynamic elements of replication protein A at the crossroads of DNA replication, recombination, and repair | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 55 | issue = 5 | pages = 482–507 | date = October 2020 | pmid = 32856505 | pmc = 7821911 | doi = 10.1080/10409238.2020.1813070 }} It also plays roles in responding to damaged DNA.
Structure
RPA is a heterotrimer, composed of the subunits RPA1 (RPA70) (70kDa subunit), RPA2 (RPA32) (32kDa subunit) and RPA3 (RPA14) (14kDa subunit). The three RPA subunits contain six OB-folds (oligonucleotide/oligosaccharide binding), with DNA-binding domains (DBD) designated DBDs A-F, that bind RPA to single-stranded DNA.
DBDs A, B, C and F are located on RPA1, DBD D is located on RPA2, and DBD E is located on RPA3. DBDs C, D, and E make up the trimerization core of the protein with flexible linker regions connecting them all together. Due to these flexible linker regions RPA is considered highly flexible and this supports the dynamic binding that RPA is able to achieve. Because of this dynamic binding, RPA is also capable of different conformations that leads to varied numbers of nucleotides that it can engage.
DBDs A, B, C and D are the sites that are involved in ssDNA binding.{{cite journal | vauthors = Dueva R, Iliakis G | title = Replication protein A: a multifunctional protein with roles in DNA replication, repair and beyond | journal = NAR Cancer | volume = 2 | issue = 3 | pages = zcaa022 | date = September 2020 | pmid = 34316690 | pmc = 8210275 | doi = 10.1093/narcan/zcaa022 }} Protein-protein interactions between RPA and other proteins happen at the N-terminal of RPA1, specifically DBD F, along with the C-terminal of RPA2. Phosphorylation of RPA takes place at the N-terminus of RPA2.
RPA shares many features with the CST complex heterotrimer, although RPA has a more uniform 1:1:1 stoichiometry.{{cite journal | vauthors = Lue NF, Zhou R, Chico L, Mao N, Steinberg-Neifach O, Ha T | title = The telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures | journal = PLOS Genetics | volume = 9 | issue = 1 | pages = e1003145 | year = 2013 | pmid = 23300477 | pmc = 3536697 | doi = 10.1371/journal.pgen.1003145 | doi-access = free }}
Functions
During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures. It also helps protect the ssDNA from being attacked by endonucleases. This keeps DNA unwound for the polymerase to replicate it. RPA also binds to ssDNA during the initial phase of homologous recombination, an important process in DNA repair and prophase I of meiosis.
RPA has a key role in the maintenance of the recombination checkpoint during meiosis of the yeast Saccharomyces cerevisiae.{{cite journal |vauthors=Sampathkumar A, Zhong C, Tang Y, Fujita Y, Ito M, Shinohara A |title=Replication protein-A, RPA, plays a pivotal role in the maintenance of recombination checkpoint in yeast meiosis |journal=Sci Rep |volume=14 |issue=1 |pages=9550 |date=April 2024 |pmid=38664461 |pmc=11045724 |doi=10.1038/s41598-024-60082-x |url=}} RPA appears to act as a sensor of single-strand DNA for the activation of the meiotic DNA damage response.
Hypersensitivity to DNA damaging agents can be caused by mutations in the RPA gene.{{cite journal | vauthors = Li X, Heyer WD | title = Homologous recombination in DNA repair and DNA damage tolerance | journal = Cell Research | volume = 18 | issue = 1 | pages = 99–113 | date = January 2008 | pmid = 18166982 | pmc = 3087377 | doi = 10.1038/cr.2008.1 }} Like its role in DNA replication, this keeps ssDNA from binding to itself (self-complementizing) so that the resulting nucleoprotein filament can then be bound by Rad51 and its cofactors.
RPA also binds to DNA during the nucleotide excision repair process. This binding stabilizes the repair complex during the repair process. A bacterial homolog is called single-strand binding protein (SSB).