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ALAN S. WALDMAN, Ph.D.
Smith
JA, Waldman BC, Waldman AS. A role for DNA mismatch repair
protein Msh2 in error-prone double-strand-break repair in
mammalian chromosomes. Genetics. 2005 May;170(1):355-63
We examined error-prone nonhomologous end
joining (NHEJ) in Msh2-deficient and wild-type Chinese
hamster ovary cell lines. A DNA substrate containing a
thymidine kinase (tk) gene fused to a neomycin-resistance
(neo) gene was stably integrated into cells. The fusion gene
was rendered nonfunctional due to a 22-bp oligonucleotide
insertion, which included the 18-bp I-SceI endonuclease
recognition site, within the tk portion of the fusion gene.
A double-strand break (DSB) was induced by transiently
expressing the I-SceI endonuclease, and deletions or
insertions that restored the tk-neo fusion gene's reading
frame were recovered by selecting for G418-resistant
colonies. Overall, neither the frequency of recovery of
G418-resistant colonies nor the sizes of NHEJ-associated
deletions were substantially different for the mutant vs.
wild-type cell lines. However, we did observe greater usage
of terminal microhomology among NHEJ events recovered from
wild-type cells as compared to Msh2 mutants. Our results
suggest that Msh2 influences error-prone NHEJ repair at the
step of pairing of terminal DNA tails. We also report the
recovery from both wild-type and Msh2-deficient cells of an
unusual class of NHEJ events associated with multiple
deletion intervals, and we discuss a possible mechanism for
the generation of these "discontinuous deletions."
Waldman, A.S., editor (2004) Genetic
Recombination: Reviews and Protocols, Methods in Molecular
Biology Series, vol. 262, Human Press, Totowa, NJ
No abstract available.
Bannister LA, Waldman BC, Waldman AS.
Modulation of error-prone double-strand break repair in
mammalian chromosomes by DNA mismatch repair protein Mlh1.
DNA Repair (Amst). 2004 May 4;3(5):465-74
We assayed error-prone double-strand
break (DSB) repair in wild-type and isogenic Mlh1-null mouse
embryonic fibroblasts containing a stably integrated DSB
repair substrate. The substrate contained a thymidine kinase
(tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo
fusion gene was disrupted in the tk portion by a 22bp
oligonucleotide containing the 18 bp recognition site for
endonuclease I-SceI. Following DSB-induction by transient
expression of I-SceI endonuclease, cells that repaired the
DSB by error-prone nonhomologous end-joining (NHEJ) and
restored the correct reading frame to the tk-neo fusion gene
were recovered by selecting for G418-resistant clones. The
number of G418-resistant clones induced by I-SceI expression
did not differ significantly between wild-type and
Mlh1-deficient cells. While most DSB repair events were
consistent with simple NHEJ in both wild-type and
Mlh1-deficient cells, complex repair events were more common
in wild-type cells. Furthermore, genomic deletions
associated with NHEJ events were strikingly larger in
wild-type versus Mlh1-deficient cells. Additional
experiments revealed that the stable transfection efficiency
of Mlh1-null cells is higher than that of wild-type cells.
Collectively, our results suggest that Mlh1 modulates
error-prone NHEJ by inhibiting the annealing of DNA ends
containing noncomplementary base pairs or by promoting the
annealing of microhomologies.
Smith JA, Waldman AS. Determination of
intrachromosomal recombination rates in cultured mammalian
cells. Methods Mol Biol. 2004;262:13-23
Recombination is involved in many
important biological processes including DNA repair, gene
expression, and generation of genetic diversity.
Recombination must be carefully regulated so as to prevent
the deleterious consequences that may result from
rearrangements between dissimilar sequences in a genome. It
is of considerable interest to study the mechanisms by which
genetic rearrangements in mammalian chromosomes are
regulated in order to understand better how genomic
integrity is normally maintained and to gain insight into
the types of genetic mutations that may destabilize the
genome. To explore such issues in mammalian chromosomes, a
suitable experimental system must be developed. In this
chapter, we describe a model system for studying
intrachromosomal recombination in cultured mammalian cells.
We discuss two model recombination substrates, a method for
stably introducing the substrates into cultured Chinese
hamster ovary cells, and a method for determining rates of
intrachromosomal recombination between sequences contained
within the integrated substrates. The general approach
described here should be applicable to the study of a
variety of aspects of recombination in virtually any
cultured mammalian cell line.
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