The Escherichia coliMutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity

Abstract

All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. The methyl-directed mismatch repair pathway in Escherichia coli functions to correct DNA biosynthetic errors that arise during chromosomal replication and to discourage recombination between substantially diverged DNA sequences (1). Inactivation of the mismatch repair system results in elevated spontaneous mutation rates (2). The pathway has been reconstituted in vitro and involves the action of eight proteins (3).Initiation of mismatch repair requires MutS, MutL, and MutH in addition to a DNA mismatch, ATP, and Mg 2ϩ , and results in the generation of a nick in the unmethylated (nascent) strand of a nearby hemimethylated d(GATC) sequence (4). The transient hemimethylated state of d(GATC) sequences after replication serves as a signal to direct repair to the nascent DNA strand (5, 6). MutS recognizes and binds the mismatched base (7,8). MutL binds the MutS-mismatch complex (9), and MutH is stimulated to catalyze the endonucleolytic cleavage at the d(GATC) site in the presence of MutL and MutS (4). After the initiation stage of mismatch repair, DNA unwinding is initiated at the nick by DNA helicase II (UvrD) and proceeds to a point beyond the error (10, 11). Excision of the error-containing DNA strand is facilitated by the action of one of several exonucleases (depending on the polarity of the reaction) which serve to degrade the single-stranded DNA (ssDNA) 1 as it is unwound by UvrD (11, 12). In the presence of ssDNA-binding protein, DNA polymerase III holoenzyme catalyzes repair synthesis on the resulting gapped DNA molecule to restore the correct sequence, and DNA ligase seals the final nick (3).The E. coli MutH protein possesses a weak endonuclease activity that is specific for unmethylated d(GATC) sequences (13). In the presence of ATP, MutS, MutL, and a hemimethylated DNA substrate containing a mismatched base pair, the MutH-associated endonuclease activity is greatly stimulated (4). However, the mechanism by which the MutH endonuclease activity is activated by the MutS-MutL complex is not known.Recently, we identified a physical interaction between the MutL and UvrD proteins using a yeast two-hybrid screen with UvrD as bait (14). Simultaneously, a biochemical interaction was reported between MutL and UvrD (15). To identify other potential interactions involving E. coli mismatch repair proteins, all possible pairwise combinations of MutS, MutL, MutH, and UvrD were tested for interactions using the yeast twohybrid system. An interaction was identified between MutL and MutH which was subsequently confirmed by affinity chromatography. The weak endonuclease activity of MutH on unmethylated d(GATC) sequences was greatly stimulated b...

Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

“…5A). Quantitation of MutH cleavage at the hemimethylated GATC site in the absence of MutS revealed that 20 -30% of the substrates were cleaved after 1 h reflecting low level activation of MutH by MutL alone as reported previously (11,25,26). In the presence of MutS, cleavage at the hemimethylated site by MutH approached 65-70% after 1 h (Fig.…”

“…In either case, MutS remains near the mismatch and can continue to specify the location of the mismatch to other participants. Activation of MutH for cleavage at a hemimethylated GATC site can occur via protein-protein interactions mediated by MutL (26,31). Implicit in this model is the formation of a looped DNA intermediate during activation of MutH by MutS and MutL that may be related to the ␣-loop structures previously observed by electron microscopy (8).…”

Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

“…In fact, recent analysis of human MutL␣ has suggested that the E. coli and human mismatch repair reactions may differ significantly in this regard. In contrast to E. coli MutL, which is believed to couple mismatch recognition by MutS to the activation of downstream activities including the excision system (10,30), human MutL␣ has been shown to be a latent endonuclease that is activated in a mismatch-, MutS␣-, replication factor C-, and proliferating cell nuclear antigen-dependent manner (31). Attempts to detect endonuclease activity associated with E. respectively, thus yielding a GAATTC site.…”

A conserved MutS homolog connector domain interface interacts with MutL homologs

“…This complex then recruits the MutL homodimer in E. coli or, in eukaryotes, one of two MutL heterodimeric complexes, Mlh1-Pms1 or Mlh1-Mlh3, in an ATP-dependent manner (12)(13)(14)(15)(16). In E. coli, MutS-MutL-DNA ternary complex stimulates the endonucleolytic activity of MutH, which makes single-strand breaks in the unmethylated DNA strand at transiently hemimethylated GATC sites and thus distinguishes the unmethylated daughter DNA strand from the methylated parental DNA strand during and after DNA replication (17)(18)(19). The nick serves to mediate excision and strand resynthesis of the newly synthesized DNA to remove the mispair (20)(21)(22).…”