hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci

Abstract

Proliferating cell nuclear antigen (PCNA) has been implicated in eukaryotic postreplicative mismatch correction, but the nature of its interaction with the repair machinery remained enigmatic. We now show that PCNA binds to the human mismatch binding factors hMutS␣ and hMutS␤ via their hMSH6 and hMSH3 subunits, respectively. The N-terminal domains of both proteins contain the highly conserved PCNA-binding motif Qxx[LI]xx [FF]. A variant of hMutS␣, lacking this motif because of deletion of 77 N-terminal residues of the hMSH6 subunit, no longer was able to interact with PCNA in vitro and failed to restore mismatch repair in hMSH6-deficient cells. Colocalization of PCNA and hMSH6 or hMSH3 to replication foci implies an intimate link between replication and mismatch correction. We postulate that PCNA plays a role in repair initiation by guiding the mismatch repair proteins to free termini in the newly replicated DNA strands. Mismatches introduced into DNA during replication are addressed by the postreplicative mismatch repair (MMR) system. In Escherichia coli, binding of the mismatch by the MutS protein, which is able to recognize both basebase mismatches (Jiricny et al. 1988;Su et al. 1988) and insertion-deletion loops (IDLs) containing up to four extrahelical nucleotides (Parker and Marinus 1992), triggers an ATP-driven assembly of the MMR repairosome. This contains, in addition to the homodimeric MutS protein, also the strand-discrimination endonuclease MutH and the MutL homodimer, thought to play a bridging role between MutS and MutH. The process also requires the DNA helicase UvrD, single-strand DNA-binding protein Ssb, one of several exonucleases, DNA polymerase III holoenzyme, and DNA ligase. The Dam methylase, which modifies GATC sites in E. coli DNA, plays a key accessory role in the MMR process. Because this enzyme lags behind the replication fork by ∼2 min (Barras and Marinus 1989), the newly synthesized strand remains unmethylated during this time and therefore can be distinguished from the methylated, template DNA. This property is used by the MutH endonuclease, which initiates the repair process by incising the newly synthesized, unmethylated strand of the DNA heteroduplex 5Ј from an unmethylated GATC sequence. The MutL protein then loads the helicase-exonuclease complex at this site (Dao and Modrich 1998), and the error-containing strand is degraded until the mispair is eliminated. The repair tract, stabilized by the Ssb protein, is filled in by DNA polymerase III, and ligation of the remaining nick and modification of the hemimethylated DNA by Dam methylase completes the repair process (for review, see Modrich 1989Modrich , 1991Modrich and Lahue 1996).The MMR process is highly conserved throughout evolution. In eukaryotes, the mismatch recognition function is fulfilled by two heterodimeric factors composed of MutS homologs MSH2 and MSH6 (MutS␣) or MSH2

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase

“…The other reason might be that the filling of a gap opposite a 6Me G residue may not be trivial. Thus, proliferating cell nuclear antigen (PCNA)-dependent polymerases tested to date would generate 6Me G/C or 6Me G/T mispairs (Reha-Krantz et al 1996), which would be again addressed by the MMR system, because of its ability to interact with the processivity factor (Kleczkowska et al 2001). Other polymerases might have problems extending from the non-Watson-Crick 6Me G/C or 6Me G/T structures, in which case the DNA synthesis would stall at the mispairs because of the activation of the 3Ј → 5Ј proofreading activity (Khare and Eckert 2001).…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…While a role for PCNA in the gap filling step of MMR catalysed by polδ was expected, early studies revealed a surprising role for PCNA in MMR prior to gap synthesis . Conserved PIP boxes at the N-termini of both MSH3 and MSH6 (Qxxhxxaa, where x is any residue, h is hydrophobic and a is aromatic) mediate the interaction of MutSα and MutSβ with PCNA; mutation of the PIP site diminishes MMR in vivo (Bowers et al, 2001;Clark et al, 2000;Flores-Rozas et al, 2000;Gu et al, 1998;Johnson et al, 1996;Kleczkowska et al, 2001). The interaction between MMR proteins and clamp proteins is also observed in prokaryotes.…”

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

“…Eukaryotic homologues of MutL are essential for mismatch repair, but no MutH homologue is known; in fact, DNA methylation does not appear to factor in strand discrimination. One hypothesis in current literature is that the processivity clamp, PCNA, that tethers replicative polymerase δ (and ε) to primer-template DNA junctions may aid strand discrimination and possibly direct repair proteins to the 3′-hydroxyl terminus of the new DNA (5). Proofreading exonucleases (or as yet unknown exonucleases) could then excise DNA beyond the mismatch, followed by polymerase δ and/or ε-catalyzed DNA synthesis and ligation of the nick-likely by DNA ligase I (6,7); ExoI, a 5′-3′ exonuclease that binds repair proteins, may also participate in mismatch excision (8).…”