Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
OF BIOLOGICAL CHEMISTRY
Vol. 268, No. 16, Issue of June 5, pp. 11823-11829,1993
Printed in U.S.A.
Methyl-directed Mismatch Repair Is Bidirectional*
(Received for publication, January 15, 1993)
Deani L. Cooper, Robert S. Lahue$, and Paul Modrichg
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Methyl-directed mismatch repair is initiated by the
mismatch-provoked, MutHLS-dependent cleavage of
the unmodified strand at a hemimethylated d(GATC)
sequence. This reaction is independent of the polarity
of the unmodified strand and can occur either 3’ or 5’
to the mismatch on the unmethylated strand (Au, K.
G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem.
The overall repair reaction also
occurs without regard to polarity
strand. Both hemimethylated configurations
heteroduplex containing a single d(GATC) sequence
are subject to methyl-directed correction in Esche-
richia coli extracts and in a purified repair system.
Repair of both heteroduplex orientations requires
MutH, MutL, MutS, DNA helicase 11, SSB, and DNA
polymerase 111 holoenzyme, but the two substrates
fer with respect to exonuclease requirements for cor-
rection. When the unmethylated d(GATC) sequence
that directs repair is located 5’ to the mismatch on the
unmodified strand, mismatch correction requires the
6’ 4 3’ hydrolytic activity of exonuclease VI1 or RecJ
exonuclease. Repair directed by
d(GATC) sequence situated 3’ to the mismatch depends
on the 3’ 4 5‘ activity of exonuclease I. Specific re-
quirements for these activities are evident with circu-
lar heteroduplexes containing a single asymmetrically
placed d(GATC) sequence, with the requirement for a
6’ 4 3’ or 3‘ 4 5’ hydrolytic activity being determined
by the orientation of the unmethylated strand along
the shorter path joining the two
This observation suggests that the methyl-directed re-
pair system utilizes the proximal
To our knowledge, these
ments represent the first instance in
I, exonuclease VII, and RecJ have been implicated in a
particular DNA metabolic pathway.
of the unmethylated
of a linear
sites in the DNA circle.
d(GATC) sequence to
Mismatched base pairs that result from replication errors
in Escherichia coli are corrected by a strand-specific pathway
in which repair is targeted to the daughter strand by the
transient undermethylation at d(GATC) sites in newly syn-
thesized DNA (reviewed in Refs. 1-3). This repair system has
a broad mismatch specificity, and a d(GATC) site can direct
repair of a mismatch at a distance of a kilobase or more (4,
5). A methyl-directed reaction, which reproduces the substrate
* This work was supported by Grant GM23719 from the National
Institute of General Medical Sciences. The costs of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
3 Present address: Dept. of Biochemistry and Molecular Biology,
University of Massachusetts Medical Center, 55 Lake Ave. North,
Worcester, MA 01655.
3 TO whom correspondence and reprint requests should be ad-
dressed. Tel.: 919-684-2775; Fax: 919-684-8885.
specificities observed in vivo and in cell-free extracts, has
been reconstituted in a purified system comprised of E. coli
MutH, MutL, MutS, DNA helicase 11, single-strand DNA
binding protein (SSB),’ DNA polymerase I11 holoenzyme,
DNA ligase, and exonuclease I (6). Initiation of repair by this
system involves the mismatch-provoked activation of a
MutH-associated d(GATC) endonuclease in a reaction that
requires MutS and MutL and is dependent upon ATP hy-
drolysis (7, 8). Incision of the unmethylated strand by this
activated d(GATC) endonuclease provides a single strand
break that suffices to target repair to the unmodified strand
(6, 9, 10).
Analysis of the MutHLS initiation reaction demonstrated
that d(GATC) cleavage by activated MutH can occur either
3’ or 5‘ to the mismatch on the unmodified strand of hemi-
methylated DNA (8). Mismatch-d(GATC) interaction during
initiation of a repair event therefore occurs without regard to
heteroduplex orientation. The experiments described here
demonstrate that heteroduplex repair can in fact be directed
by a d(GATC) sequence located either 3’ or 5’ to the mis-
match, a finding consistent with
this might be the case (8, 11, 12). With the exception of
required exonucleases, the same set of proteins is required for
correction of both heteroduplex orientations. A 3’ + 5‘ exo-
nuclease is required for repair when the unmethylated
previous suggestions that
d(GATC) sequence resides 3‘ to the mismatch, and a 5’ - 3’
activity is necessary for correction of the alternate orienta-
tion. These observations indicate
system is capable of bidirectional action. The accompanying
manuscripts (13, 14) describe the nature of excision tracts
produced by this bidirectional repair system and demonstrate
that the human strand-specific pathway shares a similar
bidirectional excision capability.
that the methyl-directed
MATERIALS AND METHODS
E. coli and Phage Strains-E. coli K-12 strains used in this work
are listed in Table I. DLC310 was constructed by P1 transduction
(15) of recJ284::TnIO from JC12123 (16) into KLC381 (17) with
selection for tetracycline resistance. The presence of rec&TnlO in
DLC3lO was confirmed by transduction of tetracycline resistance
into JC7623. The resulting isolate (DLC231) displayed the extreme
UV-sensitivity expected for a recJ recB recC sbcB genotype (16).
Exonuclease I deficiency of sbcB strains was confirmed by exonuclease
assay of crude extracts by a method described below.
Phage flMR19, which contains an EcoRI site at position 2728, was
derived from phage flMR3 (18) by introduction of a silent T to C
mutation at position 2733 using oligonucleotide methods (19). Phage
flMR20 was constructed by exchange of the 502-bp NheI-AccI frag-
ment (coordinates 5621-6123) of flMRl (18) with the corresponding
fragment of flMR19. These constructions were confirmed by dideoxy
sequence analysis of appropriate regions of the DNAs.
Proteins-A near-homogeneous sample of RecJ exonuclease was
kindly provided by R. Kolodner (Dana-Farber Cancer Institute),
DNA polymerase I and T4 DNA ligase were gifts from W. Brown
‘The abbreviations used are: SSB, single-strand DNA binding
protein; bp, base pair(s).
Bidirectional Mismatch Repair
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