Human Exonuclease I Is Required for 5′ and 3′ Mismatch Repair

Abstract

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI copurifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5 to 3 hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5 or 3 to the mispair. When the strand break that directs repair is located 3 to the mispair, hEXOI-and mismatch-dependent gap formation in excision-depleted extracts requires both hMutS␣ and hMutL␣. However, excision directed by a 5 strand break requires hMutS␣ but can occur in absence of hMutL␣. In systems comprised of pure components, the 5 to 3 hydrolytic activity of hEXOI is activated by hMutS␣ in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5-heteroduplex correction. The involvement of hEXOI in 3-heteroduplex repair suggests that it has a regulatory/ structural role in assembly of the 3-excision complex or that the protein possesses a cryptic 3 to 5 hydrolytic activity.Human cells possess a strand-specific mismatch repair system that is similar to that of Escherichia coli and depends on structural and functional homologs of bacterial MutS and MutL (1-4). Inactivation of genes that encode the mammalian MutS homologs MSH2 or MSH6 or the MutL homologs MLH1 or PMS2 confers genetic instability and a predisposition to tumor development. Eleven activities have been implicated in E. coli methyl-directed mismatch repair, and the reaction has been reconstituted in a pure system (5-8). However, our understanding of the reaction in higher cells is limited.Analysis of the human reaction in nuclear extracts, using model heteroduplexes in which a strand-specific single strand break directs repair to the incised DNA strand (9, 10), has indicated that the reaction occurs in several steps by a mechanism similar to that of E. coli mismatch correction (11,12). Repair is initiated via mismatch recognition by hMutS␣ (the hMSH2⅐hMSH6 heterodimer) (13,14) or hMutS␤ (hMSH2⅐ hMSH3 heterodimer) (15-17). hMutL␣ (hMLH1⅐hPMS2 heterodimer) and PCNA 1 are also required during the earliest stages of the reaction since inactivation of either of these activities blocks repair at or prior to initiation of excision (18 -20), which removes that portion of the incised strand spanning the strand break and the mispair (21,22). Subsequent repair DNA synthesis depends on DNA polymerase ␦ and PCNA (23, 24). hRPA, the human single-stranded DNA binding protein, has also been implicated in mismatch repair (25), but the stages of the reaction during which this protein functions have not been defined.The excision step of E. coli methyl-directed mismatch correction depends on DNA helicase II, as well as several 3Ј to 5Ј and 5Ј to 3Ј exonucleases that display specificity for single-stranded DNA (6 -8, 26). Several activities have b...

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…Eukaryotic MMR requires not only the well-characterised MSH (MutS homologue) proteins which survey the genome for both mismatches and small insertion/deletion loops but also exonucleases such as EXO1 (see Hsieh and Yamane 2008 and references therein). Eukaryotic EXO1 is a member of the Rad2/FEN1 family of nucleases (Lee and Wilson 1999) (Table 2) and is one of four nucleases that may be involved in MMR, although it is not (as originally reported) an orthologue of the Escherichia coli MMR protein EXO1 (Genschel et al 2002;Tishkoff et al 1997Tishkoff et al , 1998. There are some reports that loss of EXO1 gives rise to one form of the MMR-deficient syndrome human nonpolyposis colon cancer, HNPCC (Wu et al 2001), but a direct association has been questioned (Thompson et al 2004).…”

“…It can also degrade RNA in vitro. EXO1 is also involved in 3′-nick directed repair, suggesting either that it might have a cryptic 3′-5′ activity or that it is required to activate another as yet uncharacterised 3′-5′ nuclease (Genschel et al 2002). EXO1 has been shown to resect DNA in vitro, with DNA affinity increased by the RecQ helicase BLM and loading and processivity by the MRN complex and RPA (Nimonkar et al 2011).…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…A second puzzle is the absence of a requirement for a 3′-5′ exonuclease in reconstituted 3′-nick-directed MMR reactions and a surprising requirement for EXO1, a 5′-3′ exonuclease, in the 3′-directed reaction (Genschel et al, 2002;Dzantiev et al, 2004). How is the excision step being carried out in this case?…”

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

“…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).…”