Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture)

Abstract

DNA molecules are not completely stable, they are subject to chemical or photochemical damage and errors that occur during DNA replication resulting in mismatched base pairs. Through mechanistic studies Paul Modrich showed how replication errors are corrected by strand-directed mismatch repair in Escherichia coli and human cells.

Keywords: DNA repair; Nobel lecture; endonucleases; mismatch.

FIGURE 1

E. coli methyl-directed mismatch repair.

FIGURE 2

Methyl-directed mismatch repair in E. coli cell extracts. A. Assay for in vitro mismatch repair. Presence of the G-T mismatch within the EcoRI recognition sequence in unrepaired DNA blocks cleavage by this endonuclease. B. In vitro repair of heteroduplex shown in panel A. Residual heteroduplex repair occurring in the absence of exongenous dNTPs (right lane) is due to presence of the DNA biosynthetic precursors in the extract (18). Panel A is adapted with permission from reference ; panel B is adapted from reference .

FIGURE 3

Methyl-directed repair in E. coli extract supports bidirectional excision. Incubation of 5′ (left) or 3′ (right) hemimethylated G-T heteroduplex DNA in E. coli extract in the presence if dideoxynucleoside-5′-triphosphates results in production of a single-strand gap that spans the shorter path between the two DNA sites (18,19).

FIGURE 4

Biological activities of MutS and MutL. A. MutS binds mismatched base pairs. B. Crystal structure of the E. coli MutS dimer bound to a G-T mismatch was determined by Titia Sixma and colleagues (25). C. MutL is recruited to the MutS-mismatch complex in an ATP-dependent fashion. Panel A is reproduced from reference ; the image in panel B was provided by Titia Sixma with permission.

FIGURE 5

Methyl-directed mismatch repair in a purified system. A. The heteroduplex substrates used in these experiments contained a mismatched base pair within overlapping recognition sites for two restriction enzymes (16), which permits repair on either DNA strand to be monitored. B. Repair of a G-T heteroduplex is methyl-directed and requires presence of a hemimethylated d(GATC) site. Panels A and B are reproduced from reference with permission from AAAS.

FIGURE 6

MutH activation and initiation of methyl-directed mismatch repair.

FIGURE 7

Excision and repair synthesis steps of methyl-directed mismatch repair.

FIGURE 8

Mismatch repair of nicked heteroduplexes in human cell extracts. A. Schematic of substrate design and mechanism of repair deduced from extract experiments. B. Mismatch repair in nuclear extracts of human cells is directed to the strand that contains a preexisting strand break (N). No significant repair occurs on the covalently continuous strand (C). Panel B is reproduced from reference .

FIGURE 9

Microsatellite unstable tumor cell lines are defective in mismatch repair and deficient in MutSα or MutLα. A. Mismatch repair activity in microsatellite stable and unstable cell lines. B. Isolation of MutSα (MSH2-MSH6 heterodimer). C. Isolation of MutLα (MLH1-PMS2 heterodimer). Panel B is reproduced from reference with permission from AAAS. Panel C is reproduced from reference , Copyright 1995 National Academy of Sciences, U.S.A.

FIGURE 10

5-azacytidine exposure results in transient MLH1 expression in AN3CA tumor cells. Cells were treated with 5-azacytidine for 24 hours on days 2 and 5. Levels of MLH1 and the actin loading control were determined by western blot. The figure is reproduced with permission from reference , Copyright 1998 National Academy of Sciences, U.S.A.

FIGURE 11

MutSα activation of Exo1 and control of processive action of the MutSα-Exo1 complex by RPA.

FIGURE 12

MutLα is a strand-directed endonuclease that depends on a mismatch, a preexisting strand break, MutSα, PCNA, and RFC for activation.

FIGURE 13

Mismatch removal from MutLα-incised heteroduplex DNA by MutSα-activated Exo1 (left) or synthesis-driven strand displacement by DNA polymerase δ (right).

FIGURE 14

MutLα endonuclease active site motif. C-terminal PMS2 DQHA(X)₂E(X)₄E endonuclease active site motif is conserved in eukaryotic PMS2 homologs (S. cerevisiae PMS1 is a homolog of human PMS2) and in many bacterial MutL proteins, with the exception of MutL proteins from bacteria like E. coli that rely on d(GATC) methylation to direct mismatch repair. Amino acid residues shown in blue at the top of the figure correspond to substitution mutations used to assess involvement of the motif in MutLα function. The figure is reproduced with from reference , Copyright 2006 with permission from Elsevier.

FIGURE 15

Strand direction of MutLα endonuclease action is determined by the orientation with which the PCNA sliding clamp is loaded onto the DNA helix.