RAD51 Protein ATP Cap Regulates Nucleoprotein Filament Stability

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High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the Dloop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.

Section: Hmsh2-hmsh6 Sliding Clamp Is Constrained Within the D-loop Butmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Hmsh2-hmsh6 Sliding Clamp Is Constrained Within the D-loop Butmentioning

confidence: 99%

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Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

Honda

Okuno

Hengel

et al. 2014

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“…HsMSH2-HsMSH6 and HsRPA were purified as previously described (37,38). HsEXOI and HsMLH1-HsPMS2 were purified with modifications to published methods (SI Appendix, SI Methods) (9, 11).…”

Section: Methodsmentioning

confidence: 99%

Dynamic control of strand excision during human DNA mismatch repair

Jeon

Kim

Martín-López

et al. 2016

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Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3′-or 5′-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR.is a highly conserved strand-specific excision-resynthesis process that corrects nucleotide misincorporation errors during replication and nucleotide mismatches arising from recombination between heteroallelic parents or physical damage to the DNA (for review see ref. 1). Mutation of core MMR components results in elevated mutation rates and susceptibility to a variety of cancers (2).MMR has been reconstituted with purified Escherichia coli, Saccharomyces cerevisae, and human proteins (3-6). The core MutS homologs (MSH) and MutL homologs (MLH/PMS) components direct a strand-specific excision reaction, whereas resynthesis appears to be uniquely performed by the replicative polymerase complex (1). In all organisms the excision process is initiated at a single-strand DNA scission (ssDNA/S) that may be located either 3′ or 5′ and hundreds to thousands of base pairs distant from the mismatch (4, 7). An ssDNA/S positioned on the newly replicated strand ensures accurate correction of replication misincorporation errors (1).Excision directionality in γ-proteobacteria (E. coli) is linked to the choice of 3′ or 5′ exonucleases that specifically degrade ssDNA generated by the EcUvrD helicase in concert with EcMutS and EcMutL (1). The lack of a helicase distinguishes yeast and human MMR from γ-proteobacteria. Moreover, the eukaryotic 3′-and 5′-excision reactions require different core MMR components and likely occur by different mechanisms (1). For example, the 3′-MMR excision requires the replicative processivity factor PCNA to activate a cryptic MLH/PMS endonuclease activity (8), whereas 5′ MMR uses the only known MMR exonuclease EXOI (3, 5, 6). Unlike the E. coli ssDNA exonucleases, EXOI will initiate 5′ excision from a ssDNA/S in the absence of a helicase (9). Whereas the purified 5′-MMR reaction does not require MLH/PMS or PCNA, complementation studies with cellular extracts displayed a substantial requirement for...

“…turnover (26). It was more difficult to anticipate the effect of placing an additional negative charge next to this charged aspartate.…”

Section: Significancementioning

confidence: 99%

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Subramanyam

Ismail

Bhattacharya

et al. 2016

The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-L-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.RAD51 recombinase | c-Abl tyrosine kinase | homologous recombination | single-molecule total internal reflection fluorescence microscopy | phosphorylation T he DNA in the human genome is constantly subjected to damage. This damage is a byproduct of normal cellular metabolism, exposure to radiation, and environmental mutagens (1). Homologous recombination (HR) and the pathways that use the machinery of HR are responsible for the accurate repair of the most deleterious DNA lesions, including double-stranded DNA breaks (DSBs), interstrand DNA cross-links, and damaged replication forks, and thereby contribute to maintenance of the stable genome (2-5). HR also plays an important role in telomere maintenance (6). HR, a process highly conserved throughout evolution, is carried out through a precisely coordinated and tightly regulated series of events. The key step in HR is the assembly of a RecA-family recombinase (phage UvsX, bacterial RecA, archaeal RadA, or eukaryotic RAD51) onto resected single-stranded DNA (ssDNA). The recombinase forms a nucleoprotein filament that then invades homologous duplex DNA, resulting in a displacement loop structure that can be used as a primer for synthesis using the intact duplex as a template. Similar to its bacterial and yeast homologs, the human RAD51 binds ssDNA in an ATP-dependent manner (7). The nucleoprotein filament formed by the ATP-bound RAD51 is arranged such that each RAD51 monomer binds three nucleotides, forming the pairing unit in these reactions (8). Beyond these basic attributes, the characteristics of human RAD51 protein differ significantly from its archaeal, bacterial, and yeast homologs.The critical role of HR requires all steps of this process ...