PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

Abstract

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.MutLalpha | genetic instability | cancer | triplet repeats

Acetylation regulates DNA repair mechanisms in human cells

“…HCT116 has a defective MLH1 gene expressing a component of MutLa, an endonuclease directed by PCNA to perform incision near a mismatch. 33 In our system, nicks are already present in the plasmid construct. MLH1 is also involved in recruitment of EXO1.…”

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

“…endonuclease is activated in a mispair-dependent fashion by a combination of Msh2-Msh6 or Msh2-Msh3, PCNA, and RFC to generate DNA nicks 5′ to the mispair (29)(30)(31)(32)(33). Once the 5′ nicks are formed, repair appears to occur as observed in the 5′ nickdirected MMR reactions.…”

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

“…In this constellation, its complex with PCNA would always nick the red (5'3') strand, irrespective of its distance from the PCNA loading site, or its rotation around the helix axis. Surprisingly, the above study [16] showed that RFC can load PCNA with low efficiency also onto DNA lacking pre-existing nicks or gaps (e.g. supercoiled substrates, or molecules containing bubbles or stem-loops).…”

“…It seems likely that expansions arise from structures (cruciforms, stem-loops/loops) containing full-length TNRs in both strands, which these repetitive sequences have a propensity to form. Assuming that, as mentioned above, RFC were to load PCNA at these structures [16], then its association with MutSβ and MutLα might result in an endonucleolytic cleavage of either strand ( Figure 2B), which would cause collapse of the structure and generate a 3' terminus that could prime repair synthesis ( Figure 2C). A subset of the cleaved structures could give rise to heteroduplexes with one strand containing a large insertion (Figure 2D), the repeated processing of which by the MMR proteins and repair polymerase(s) would generate an expanded TNR.…”

“…How MutLα introduces additional breaks selectively into the nicked strand was elucidated in the follow-up study [16], which showed that the MutLα endonuclease is activated by interaction with PCNA. This homotrimeric ring, which has two distinct faces, is loaded on the DNA by the clamploader RFC, mostly at boundaries between single-and double-stranded DNA [17], and always with the same face oriented towards the 3'terminus (shown light brown in Fig.…”