DNA Repair Protocols: Mammalian Systems

517 Pages·2006·4.65 MB·English

by  Malgorzata Z. Zdzienicka (auth.)

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M E T H O D S I N M O L E C U L A R B I O L O G Y™
DNA Repair Protocols
Mammalian Systems
S E
ECOND DITION
Edited by
Daryl S. Henderson
Pharmacological Sciences, SUNY Stony Brook
Stony Brook, NY
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Cover illustration: Artistic rendering of a confocal microscope image of human fibroblasts irradiated with
ultraviolet light through a micropore filter, immunostained with antibodies to PCNA and CAF-1, and coun-
terstained with Hoechst 33258 to reveal DNA. Original image courtesy of Dr. Ennio Prosperi. See Chapter
31, "Analysis of Proliferating Cell Nuclear Antigen (PCNA) Associated with DNA Excision Repair Sites in
Mammalian Cells," by A. Ivana Scovassi and Ennio Prosperi.
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Library of Congress Cataloging in Publication Data
DNA repair protocols : mammalian systems / edited by Daryl S. Henderson.—2nd ed.
p. ; cm. — (Methods in molecular biology ; 314)
Includes bibliographical references and index.
ISBN 1-58829-513-3 (alk. paper) EISBN 1-59259-973-7
1. DNA repair—Laboratory manuals.
[DNLM: 1. DNA Repair—Laboratory Manuals. 2. DNA Damage—Laboratory Manuals. 3.
Mammals—genetics—Laboratory Manuals. QU 25 D755 2005] I.
Henderson, Daryl S. II. Series: Methods in molecular biology (Clifton, N.J.); v. 314.
QH467.D627 2005
572.8’6—dc22 2005006250
Preface
The first edition of this book, published in 1999 and called DNA Repair
Protocols: Eukaryotic Systems, brought together laboratory-based methods for
studying DNA damage and repair in diverse eukaryotes: namely, two kinds of
yeast, a nematode, a fruit fly, a toad, three different plants, and human and murine
cells. This second edition of DNA Repair Protocols covers mammalian cells only
and hence its new subtitle, Mammalian Systems. There are two reasons for this
fresh emphasis, both of them pragmatic: to cater to the interests of what is now a
largely mammalocentric DNA repair field, and to expedite editing and produc-
tion of this volume.
Although DNA Repair Protocols: Mammalian Systems is a smaller book
than its predecessor, it actually contains a greater variety of methods. Fourteen
of the book’s thirty-two chapters are entirely new and areas of redundancy
present in the first edition have been eliminated here (for example, now just two
chapters describe assays for nucleotide excision repair [NER], rather than
seven). All eighteen returning chapters have been revised, many of them exten-
sively. In order to maintain a coherent arrangement of topics, the four-part par-
titioning seen in the first edition was dispensed with and chapters concerned
with ionizing radiation damage and DNA strand breakage and repair were relo-
cated to near the front of the book. Finally, an abstract now heads each chapter.
I have aimed to make DNA Repair Protocols: Mammalian Systems a well-
rounded book, intended to address a broad range of questions about practical
mammalian DNA repair, including arcana such as “what is radioresistant DNA
synthesis and how is it measured?” (see Chapter 5, by Jaspers and Zdzienicka).
The final selection of topics was influenced by both the contents of the first
edition (naturally) and the willingness of its authors to contribute again; by my
desire to correct deficiencies in the first edition (e.g., it lacks a chapter on DNA
helicase assays); and by what new methods have come into use since 1998,
when the first edition went into production. Below I summarize and put into
context some of what’s new, as a way of illustrating the diversity and scope of
DNA Repair Protocols: Mammalian Systems. (My apologies to authors of chap-
ters not mentioned; no slight is intended.)
Cytogenetic analysis, a topic that had scant coverage in the first edition, is
emphasized in Chapters 3 and 4. In Chapter 3, Au and Salama detail a cytoge-
netic challenge assay in which blood lymphocytes obtained from individuals of
interest (e.g., smokers, chemical workers, chemotherapy patients) are irradi-
ated—that is, challenged—with ionizing or UV radiation and then scored for
v
vi Preface
chromosomal anomalies. Abnormally high levels of induced chromosomal aber-
rations may be indicative of a constitutional repair deficiency, as the authors and
their colleagues recently described for certain variant alleles of two DNA repair
genes, XPD and XRCC1.
The cellular response to ionizing radiation is complex. For example, the irra-
diated cell may be killed outright or it may repair whatever damage occurs and
continue to grow and divide no worse for wear. A third outcome, only recently
appreciated, goes something like this: the cell survives irradiation apparently
unharmed and proliferates, but then its descendents go on to display “genomic
instabilities” of various kinds. This intriguing transgenerational phenomenon (or
phenomena) and cytological methods for studying it are described by Nagar,
Corcoran, and Morgan in Chapter 4.
In Chapter 7, Huang and Darzynkiewicz present a new cytometric approach
for detecting DNA double-strand breaks (DSBs). Their method is based on work
by W. M. Bonner and colleagues who discovered that a phosphorylated form of
histone H2AX, called γH2AX, accumulates in quantity at chromosomal DSBs.
Specific antibodies against γH2AX are now available commercially, making im-
munofluorescence detection of γH2AX a convenient way to detect DSBs induced
by clastogenic agents (with attendant caveats noted in the chapter).
Two very different methods for quantifying levels of DNA damage (Chap-
ters 14 and 21) both make use of PicoGreen, an ultrasensitive nucleic acid stain
that shows especially strong enhancement of fluorescence when bound to
double-stranded DNA (dsDNA). In Chapter 14, Santos, Meyer, Mandavilli and
Van Houten describe a quantitative PCR (QPCR) method that can be used to
measure DNA damage in specific regions of either the mitochondrial or nuclear
genome. The crux of this method is that DNA lesions able to impede move-
ment of the polymerase during PCR will cause less product to be synthesized
compared to control reactions using a lesion-free template; in both cases the
amount of dsDNA product is quantified with PicoGreen. The QPCR technique
combines aspects of Bohr’s pioneering Southern blot method, described by
Anson, Mason, and Bohr in Chapter 13 (both approaches quantify
underrepresentation of a specific DNA fragment and use Poisson probabilities
to calculate levels of damage), and Pfeifer’s ligation-mediated PCR in Chapter
15 (the stopping of polymerases by lesions in DNA). (Note that Hays and
Hoffman in the first edition described a QPCR approach for measuring activi-
ties of photolyases, although that method uses UV-damaged plasmids as tem-
plate and radiolabel to quantify PCR product levels.)
In Chapter 21, Schröder, Batel, Schwertner, Boreiko, and Müller detail their
sensitive microtiter plate assay (“Fast Micromethod”) for estimating levels of
single-strand breaks (nicks) in DNA. The single-strand breaks are identified
indirectly by measuring the reduction in amount of dsDNA—quantified by
Preface vii
PicoGreen—in mutagen-treated and untreated samples following alkaline dena-
turation. Nicked duplex DNA denatures more readily and hence loses fluores-
cence more rapidly than does intact DNA. The Fast Micromethod shares some
methodological features with the alkaline version of the comet assay described
by Speit and Hartmann in Chapter 20 (e.g., denaturation of DNA under alkaline
conditions). In addition, it can be adapted for high throughput screening and
allows testing of both cells and tissues, including frozen material.
Chapter 22, contributed by Shibutani, Kim, and Suzuki, describes a vastly
improved 32P-postlabeling protocol, which is a very sensitive method for detect-
ing the presence of “adducted” bases in DNA. 32P-postlabeling protocols have
traditionally used thin layer chromatography to separate modified deoxynucle-
otides from their normal counterparts. The new technique, developed in
Shibutani’s laboratory, uses polyacrylamide gel electrophoresis, which allows
multiple samples to be run out and compared on a single gel. Other strengths of
the method are its improved handling and high sensitivity.
In Chapter 24, Wang and Hays describe a new and efficient protocol for
preparing mismatch repair (MMR) plasmid substrates. Their method makes
use of nicking endonucleases that introduce a single-strand nick at specific
sequences in duplex DNA. One nicking endonuclease is used to create a pair of
nicks separated by tens of nucleotides on one strand of a plasmid specially
engineered for this purpose. The oligonucleotide defined by the nicks is melted
out by heating, the resulting gapped plasmid is purified by BND-cellulose chro-
matography, and the desired mismatch-containing synthetic oligomer is annealed
into the gap. Following ligation and purification, the mismatch-containing plas-
mid substrate is treated with a different nicking endonuclease to generate the
nick that is essential for initiation of MMR excision.
In the first edition, Matsumoto described a base excision repair (BER) assay
using Xenopus oocyte extracts. Here, in Chapter 26, he details an updated proto-
col for studying BER in mammalian cells that includes several important techni-
cal refinements. For example, in the synthetic BER substrate that he described in
the first edition, the abasic site was positioned opposite an adenine. In his new
construct the abasic site is opposite a cytosine, thus mimicking depurination of
guanine, which in vivo occurs much more frequently than loss of thymine. Two
further improvements to his assay are the use of SYBR Green (rather than radio-
label) to detect repair products and a simplified method for preparing whole-cell
extracts.
Missing from the first edition was a chapter on DNA helicase assays. This
significant lacuna has now been filled by Brosh and Sharma’s excellent contri-
bution, Chapter 28. New authors Scovassi and Prosperi have written a consid-
erably expanded chapter (Chapter 31) on proliferating cell nuclear antigen
(PCNA), in keeping with that molecule’s central role in DNA repair. Those
viii Preface
two chapters, together with one by Mello, Moggs, and Almouzni addressing
the role of chromatin in repair (Chapter 32), round out a strong collection of
BER- and NER-related chapters.
I thank all the authors for their excellent chapters and much patience; John
Walker, the series editor, for his advice; James Geronimo, who deftly guided
the book through production, and his colleagues at Humana Press; and Nadine
Henderson and other Hendersons and Tindalls for their interest and support.
Daryl S. Henderson
Contents
Preface ..............................................................................................................v
Contributors ...................................................................................................xiii
Technical Notes ............................................................................................xvii
1 Isolation of Mutagen-Sensitive Chinese Hamster Cell Lines
by Replica Plating
Malgorzata Z. Zdzienicka.....................................................................1
2 Complementation Assays Adapted for DNA Repair–Deficient
Keratinocytes
Mathilde Fréchet, Valérie Bergoglio, Odile Chevallier-Lagente,
Alain Sarasin, and Thierry Magnaldo...............................................9
3 Cytogenetic Challenge Assays for Assessment
of DNA Repair Capacities
William W. Au and Salama A. Salama................................................25
4 Evaluating the Delayed Effects of Cellular Exposure
to Ionizing Radiation
Shruti Nagar, James J. Corcoran, and William F. Morgan.................43
5 Inhibition of DNA Synthesis by Ionizing Radiation:
A Marker for an S-Phase Checkpoint
Nicolaas G. J. Jaspers and Malgorzata Z. Zdzienicka........................51
6 Analysis of Inhibition of DNA Replication in Irradiated Cells
Using the SV40-Based In Vitro Assay of DNA Replication
George Iliakis, Ya Wang, and Hong Yan Wang..................................61
7 Cytometric Assessment of Histone H2AX Phosphorylation:
A Reporter of DNA Damage
Xuan Huang and Zbigniew Darzynkiewicz........................................73
8 Detection of DNA Strand Breaks by Flow and Laser Scanning
Cytometry in Studies of Apoptosis and Cell Proliferation
(DNA Replication)
Zbigniew Darzynkiewicz, Xuan Huang, and Masaki Okafuji.............81
9 In Vitro Rejoining of Double-Strand Breaks in Genomic DNA
George Iliakis and Nge Cheong..........................................................95
10 Detection of DNA Double-Strand Breaks and Chromosome
Translocations Using Ligation-Mediated PCR and Inverse PCR
Michael J. Villalobos, Christopher J. Betti,
and Andrew T. M. Vaughan..........................................................109
ix