Molecular Analysis of DNA Rearrangements in the Immune System

223 Pages·1996·5.996 MB·English

by  Dik C. van Gent, J. Fraser McBlane (auth.), Dr. Rolf Jessberger, Dr. Michael R. Lieber (eds.)

Current Topics In
217 Microbiology
and Immunology
Editors
R.W. Compans. Atlanta/Gorgia
M. Cooper. Birmingham/Alabama· H. Koprowski.
Philadelphia/Pennsylvania . F. Melchers. Basel
M. Oldstone. La Jolla/California· S. Olsnes. Oslo
M. Potter. Bethesda/Maryland· H. Saedler. Cologne
P. K. Vogt. La Jolla/Californa . H. Wagner. Munich
Springer
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Molecular Analysis
of DNA Rearrangements
in the Immune System
Edited by R. Jessberger
and M.R. Lieber
With 43 Figures and 13 Tables
Springer
DR. ROLF JESSBERGER
Institute for Immunology
Grenzacherstr. 487
CH-4005 Basel
Switzerland
DR. MiCHAEL R. LIEBER
Division of Molecular Oncology
Department of Pathology
Washington University School of Medicine
Campus Box 8118, 660 So. Euclid Ave.
St. Louis, MO 63110
USA
Cover illustration: V(D)J recombination is one of three covalent alterations of
DNA that generate the genes encoding antigen receptors of the immune sys
tem. Class switch recombination and somatic hypermutation are two others.
All three, and related topics, are discussed in this volume.
The cover is a partial model for V(D)J recombination. The yellow circles re
present the RAG-7,-2 endonuclease. This endonuclease cleaves at hep
tamer/nonamer signal sequences that have either a 72-or 23-base pair spacer
between the heptamer and the nona mer (green and red triangles, respectively).
The endonuclease generates a hairpinned coding end and a blunt signal end.
Ku70/BO, a heterodimer, is the predominant DNA end binding protein in cells
and is thought to bind to DNA termini. In one model, Ku is the DNA binding
component of a complex that includes the DNA-dependent protein kinase,
DNA-PKcs. DNA-PKcs appears to be the defective component in the double
strand break repair complementation group XRCC7. XRCC7 also is the com
plementation group for murine SCID. Ku70 is defective in XRCC6, KuBO is
defective in XRCC5. In V(D)J recombination, the signal ends are joined to form
a signal joint. The V and J sub-exons are joined to form the coding joint,
creating a novel exon in the immune-specific repertoire armamentarium.
Cover design: Springer-Verlag Heidelberg, Design & Production
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00110.1007/978-3-642-50140-1
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Preface
The vertebrate immune system is distinctive among defense
systems of multicellular organisms. In addition to nonspecific
immunity, it generates a randomized array of millions of
antigen receptors (immunoglobulins and T-cell receptors). A
subset of these receptors are critical for binding to invading
microbes or biochemicals from them to tag the microbes for
elimination. Three site-directed DNA modification processes
are critical to this process in vertebrates. V(D)J recombination
generates the array of exons that encode the antigen binding
pockets. Recent work summarized in this volume describes
the dissection of this process at the biochemical level. The
mechanism of the reaction is now understood in considerable
detail. The proteins that catalyze many steps of the process
have now been identified by biochemical and genetic recon
stitution and by analysis of genetic mutants defective in V(D)J
recombination.
Class switch recombination is the process by which the
variable domain exon of the heavy chain is changed from IgM
to IgG, IgA. or IgE. Recent progress is described in the de
velopment of an extrachromosomal substrate assay system.
Molecular genetic analysis of the process in transgenics is
defining some of the cis sequence requirements. Biochemical
assays for defining enzymatic components are also described.
In addition to exciting progress in V(D)J recombination and
class switch recombination, one chapter describes recent pro
gress in somatic hypermutation. This is the process by which
affinity maturation is achieved, and it involves generation of
point mutations within and downstream of the variable domain
exons of the heavy and light chain genes of immunoglobulins.
All three of these systems are intriguing because of their
site-directed nature. How is targeting achieved, how are the
reactions carried out. what are the enzymes that catalyze
them, and how are these enzymes regulated? These are
among the questions examined in this monograph.
Basel R. JESSBERGER
St. Louis, Montana M.R. LIEBER
Contents
Initiation of V(D)J Recombination in a Cell-Free System
by RAG 1 and RAG2 Proteins ....................... .
DIK C. VAN GENT, J. FRASER McBLANE,
DALE A. RAMSDEN, MOSHE J. SADOFSKY,
JOANNE E. HESSE, and MARTIN GELLERT
rag-1 and rag-2: Biochemistry and Protein Interactions.. 11
DAVID G. SCHATZ and THOMAS M.J. LEU
Regulation of Recombination Activating Gene Expression
During Lymphocyte Development .................... 31
ULF GRAWUNDER, THOMAS H. WINKLER.
and FRITZ MELCHERS
The Cell Cycle and V(D)J Recombination .............. 45
STEPHEN DESIDERIO, WEEI-CHIN LIN, and ZHONG LI
Double-Strand Breaks, DNA Hairpins,
and the Mechanism of V(D)J Recombination .......... 61
SHARRI BOCKHEIM STEEN, CHENGMING ZHU,
and DAVID B. ROTH
Identification of the Catalytic Subunit
of DNA Dependent Protein Kinase as the Product
of the Mouse scid Gene. . . . . . . . . . . . . . . . . . . . . . . . . .. 79
PENNY A. JEGGO, STEPHEN P JACKSON
and GUILLERMO E. TACCIOLI
The DNA-Activated Protein Kinase - DNA-PK .......... 91
CARL W. ANDERSON and TIMOTHY H. CARTER
Role of the Ku Autoantigen in V(D)J Recombination
and Double-Strand Break Repair. . . . . . . . . . . . . . . . . . .. 113
GILBERT CHU
VIII Contents
Characterization of Chinese Hamster Cell Lines
That Are X-Ray-Sensitive,
Impaired in DNA Double-Strand Break Repair
and Defective for V(D)J Recombination .............. 133
SANG EUN LEE, DONG MiNG HE,
and ERIC A. HENDRICKSON
Identification of the XRCC4 Gene:
Complementation of the DSBR
and V(D)J Recombination Defects of XR-1 Cells ..... " 143
ZHIYING LI and FREDERICK WALT
Developmental and Molecular Regulation
of Immunoglobulin Class Switch Recombination ....... 151
MATIHIAS LORENZ and ANDREAS RADBRUCH
Transcription Targets Recombination
at Immunoglobulin Switch Sequences
in a Strand-Specific Manner ........................ 171
GREGORY A. DANIELS and MiCHAEL R. LIEBER
Biochemical Studies of Class Switch Recombination ... 191
ROLF JESSBERGER, MATIHIAS WABL,
and TILMAN BORGGREFE
Somatic Hypermutability ........................... 203
MATIHIAS WABL and CHARLES STEINBERG
Subject Index .................................... 221
List of Contributors
(Their addresses can be found at the beginning of their re
spective chapters)
ALT. F.W 143 LI,Z. 45
ANDERSON. C.W 91 Li. Z. 143
BOCKHEIM STEEN. S. 61 LIEBER, M.R. 171
BORGGREFE. T. 191 LIN.W-C. 45
CARTER. T.H. 91 LORENZ. M. 151
CHU, G, 113 McBLANE, J,F. 1
DANIELS, GA 171 MELCHERS, F. 31
DESIDERIO, S. 45 RADBRUCH,A, 151
GELLERT. M. 1 RAMSDEN. DA 1
GRAWUNDER. U. 31 ROTH, D.B, 61
HE. D.M. 133 SADOFSKY. MJ. 1
HENDRICKSON, E.A. 133 SCHATZ, D,G, 11
HESSE. J.E. 1 STEINBERG. C. 203
JACKSON. S.P. 79 TACCIOLI. G,E. 79
JEGGO. P.A. 79 VAN GENT. D.C, 1
JESSBERGER. R. 191 WABL, M. 191.203
LEE. S.E. 133 WINKLER. T.H. 31
LEU. T.M.J. 11 ZHU. C, 61
Initiation of V(D)J Recombination
in a Cell-Free System by RAG1 and RAG2 Proteins
DIK C. VAN GENT, J. FRASER McBLANE, DALE A. RAMSDEN,
MOSHE J. SADOFSKY, JOANNE E. HESSE, and MARTIN GELLERT
Introduction .......................................... .
2 Development of a Cell-Free System . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2
2.1 Generation of Double Strand Breaks in Pre-B Cell Nuclear Extracts . . . . .. 2
2.2 Cleavage by RAG1 and RAG2 Proteins . . . . . . 3
3 Outline of the V(D)J Cleavage Reaction . . . . 4
4 Recognition of Recombination Signal Sequences .. 6
5 Regulation of Cleavage . . 7
5.1 Coupled Cleavage at Two Signal Sequences ... . 7
5.2 Regulation of Cleavage Activity ........ . . ........ 8
6 A Possible Role for RAG Proteins in the Completion of the Recombination Reaction .. 9
References . . . . . . . . . . . . . . . . . . . . . . . . . 9
1 Introduction
Mature T cell receptor (TCR) and immunoglobulin (lg) genes are assembled
from separate gene segments, which are flanked by recombination signal
sequences (RSS), consisting of conserved heptamer and nonamer motifs sep
arated by a spacer region of 12 or 23 base pairs (bp). V(D)J recombination
results in a precise head-to-head ligation of two signal sequences in a signal
joint, and the imprecise joining of two coding segments in a coding joint,
which may contain additions or deletions of a few bp. The imprecise nature
of coding joints led to the hypothesis that double strand breaks (DSB) might
be intermediates in the recombination pathway. These DSB can then be pro
cessed by an exonuclease and/or by terminal deoxynucleotidyl transferase
(TdT) before formation of a coding joint.
Such broken molecules have indeed been found in lymphoid cells (RoTH
et al. 1992a). Signal ends are blunt, 5'-phosphorylated, and contain the complete
signal sequence (SCHLISSEL et al. 1993; ROTH et al. 1993). Coding ends were
initially found only in mice harboring the severe combined immunodeficiency
(scid) mutation. They terminated in a hairpin structure in which both strands
Laboratory of Molecular Biology. National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892-0540, USA
2 Dik C. van Gent et al.
were covalently coupled (RoTH et al. 1992b). These hairpin coding ends have
since also been found in a non-scid pre-B cell line, albeit at a very low level
(RAMSDEN and GELLERT 1995). They were hypothesized to be intermediates in
normal V(D)J recombination, since this would provide a plausible explanation
for the frequent observation of self-complementary (P) nucleotides: opening
of the hairpin away from the tip could produce such a sequence. Additional
evidence that broken molecules are indeed intermediates in V(D)J recombi
nation was obtained from studies on the 103/BCL-2 cell line, which contains
a temperature sensitive v-abl gene. Upon shift from the permissive (low) to
the non permissive (high) temperature, the v-abl protein is inactivated, ex
pression of RAG proteins is upregulated, and a very high level of recombination
is induced (CHEN et al. 1994). Under the non permissive conditions, coding
joints are formed readily, but DSB persist at the signal ends. When the cells
are shifted back to the permissive temperature, these signal ends go on to
form signal joints, showing that signal ends are really intermediates leading
to signal joints (RAMSDEN and GELLERT 1995).
Expression of two novel genes, RAG1 and RAG2, was found to be necess
ary and sufficient to confer V(D)J recombination activity on nonlymphoid cells
(SCHATZ et al. 1989; OETIINGER et al. 1990). Most evidence pointed toward a
role of RAG1 and RAG2 in initiation of V(D)J recombination; mice that do not
express both of these proteins do not make DSB (SCHLISSEL et al. 1993). More
over, a mutant form of RAG1 was found with highly increased sensitivity to
mutations in the heptamer and flanking coding sequence, consistent with the
hypothesis that RAG 1 protein interacts with this region (SADOFSKY et al. 1995).
Attempts to reconstitute complete recombination in a cell-free system
have not yielded any positive results, perhaps because of the large number
of proteins involved. The initial cleavage reaction was expected to require at
least the presence of the two RAG proteins, but the joining of these broken
molecules to form signal and coding joints requires at least several other
proteins involved in DSB repair (ROTH et al. 1995). It therefore seemed more
practical to reproduce only the initial step of V(D)J recombination (formation
of DSB) in a cell-free system.
2 Development of a Cell-Free System
2.1 Generation of Double Strand Breaks
in Pre-B Cell Nuclear Extracts
In order to develop a cell-free system for generation of DSB at RSS, we
searched for a very active source of recombination factors, and an efficient
technique for detection of broken DNA molecules. As the source of recom
bination factors we used the pre-B cell line 10 3/BCL-2, which had been induced