A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair

Abstract

Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5 to 3 hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutL␣-dependent and requires functional integrity of the MutL␣ endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutS␣, MutL␣, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase ␦ that supports Exo1-independent repair in vitro. Repair in this system depends on MutL␣ incision of the nicked heteroduplex strand and dNTPdependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells.cancer ͉ DNA polymerase ͉ DNA repair ͉ strand displacment

Acetylation regulates DNA repair mechanisms in human cells

“…2A). 26 Previous studies showed that hyperacetylation induced by sodium butyrate stimulates nucleotide excision repair in UVirradiated cells. 8,10,[47][48][49][50] The stimulation of DNA repair was shown to be associated with alterations in chromatin structure making damaged sites in DNA more accessible to repair enzymes.…”

“…2A). 24,26 HCT116 cells are deficient in MMR due to defective MLH1, which likely affects formation of the MMR complex and recruitment of EXO1. 24 In these cells, we expect that nicked DNA constructs with a mismatch become primary substrates for an EXO1-independent pathway ( Fig.…”

“…2A). 26 Thus, in this setting, repair of the mismatch near a nick is likely to result from Pol d -directed synthesis involving strand displacement with support of PCNA. Placement of the mismatch at various locations from the nick would largely report on effects of protein acetylation on mechanisms linked to this synthesis-driven displacement.…”

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

“…Once the 5′ nicks are formed, repair appears to occur as observed in the 5′ nickdirected MMR reactions. MMR can also occur in the absence of Exo1 (24,34,35); however, our knowledge of Exo1-independent MMR mechanisms is not as well developed as it is for Exo1-dependent MMR (19,36,37).…”

Mammalian mismatch repair: error-free or error-prone?

“…To date, no such enzyme could be identified. However, it could be shown that 5' nick-directed MMR in vitro could occur by an EXO1-independent mechanism, which involves strand displacement mediated by polymerase-δ, together with MutSα, MutLα, RPA, RFC and PCNA ( Figure 1D) [25]. Evidence from mammalian systems supporting the existence of EXO1-independent pathways in vivo is currently lacking.…”