Download Recognition and processing of damaged DNA

73
Journal of Cell Science, Supplement 19, 73-77 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Recognition and processing of damaged DNA
Tomas Lindahl
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK
SUMMARY
Base excision-repair, which is required for correction of
spontaneous hydrolytic and oxidative damage to DNA as
well as lesions inflicted by alkylating agents, is a relatively
well understood repair pathway. Mammalian factors
involved in this pathway are reviewed, with emphasis on
current uncertainties.
Most DNA replication and repair enzymes in
mammalian cell nuclei, e.g. DNA polymerases a , (3, 8, and
8, have direct counterparts in yeast. In contrast, the
abundant enzymes in mammalian cell nuclei that bind and
are activated specifically by DNA strand interruptions,
poly(ADP-ribose) polymerase and DNA-dependent protein
kinase, have not been detected in yeast; nor has p53, which
is elevated in response to DNA strand breaks. We have
found a family of four distinct DNA ligases in human cell
nuclei, whereas only a single DNA ligase has been detected
in yeast. It would appear that the cellular responses to DNA
strand breaks may differ markedly between higher and
lower eukaryotes.
THE BASE EXCISION-REPAIR PATHWAY
modifying enzyme (Klimasauskas et al., 1994) of swing-out of
the altered nucleoside moiety into an extrahelical position prior
to recognition as a substrate and glycosyl bond cleavage in a
snugly fitting pocket of the enzyme.
The patch size of replaced residues in the mammalian base
excision-repair process is heterogeneous, because a bifurcation
point in the pathway occurs after incision at an abasic site (Fig.
1). It is presently unclear how this choice between two alterna­
tive routes of excision and gap-filling is taken, and whether DNA
repair proteins actively regulate this event. Usually, the pathway
shown on the left in Fig. 1 appears predominant in human cells.
DNA polymerase p fills in the single-nucleotide gap (Dianov et
al., 1992; Singhal et al., 1995), the displaced deoxyribosephosphate (dRp) residue is excised, and ligation occurs to restore
the DNA structure. In Escherichia coli, hydrolytic removal of
dRp can be catalyzed by the RecJ protein (Dianov and Lindahl,
1994), a 5'-3' single-strand exonuclease, but the excision
function has not yet been clearly characterized in mammalian
cells. Thus, it is not known if deletion of the dRp moiety occurs
by hydrolysis or by (3-elimination - either strategy would be sat­
isfactory since the remaining strand interruption in DNA would
be identical and could be directly re-ligated, although the
structure of the removed dRp residue would be different (a
saturated vs unsaturated form of dRp). In the main pathway, the
DNA ligase III/XRCC1 complex is the best candidate to account
for the final ligation step. The minor alternative pathway (on the
right in Fig. 1) results in repair patches several nucleotides long,
and shares many features with gap-filling during lagging-strand
replication and nucleotide excision-repair.
Three main forms of DNA excision-repair have been found in
all living organisms. There is very little overlap between these
distinct pathways, so they must be considered as separate
processes. Two of them are nonessential. Nucleotide excisionrepair protects cells against ultraviolet light, and humans with
a defect in this form of repair suffer from xeroderma pig­
mentosum, a severe cancer-prone dermatological disease.
Mismatch repair is employed to correct occasional template
reading errors by DNA polymerases during replication, and
defective cells have a mutator phenotype which is associated
with inherited non-polyposis colon cancer in man. The third
pathway, base excision-repair, is required for removal of
frequent spontaneous hydrolytic and oxidative lesions in DNA.
Human cells totally defective in this pathway have not been
detected, and data on bacterial mutants strongly suggest that
such cells would be inviable (Lindahl, 1993).
A current scheme for DNA base excision-repair in
mammalian cells is shown in Fig. 1. A damaged base residue
is removed by hydrolysis of the base-sugar bond by one of five
different DNA glycosylases that recognise different lesions.
The glycosylase shown here specifically excises deaminated
cytosine residues from DNA. The resulting information-less
abasic site is recognised by a specific AP (apurinic/apyrimidinic) endonuclease that catalyzes hydrolysis of the phosphodiester bond on the immediate 5' side of the lesion. The threedimensional structures of these well-characterised DNA repair
enzymes recently have been determined at high resolution
(Savva et al., 1995; Mol et al., 1995a and 1995b), and there is
convincing genetic evidence in microorganisms to support
their assigned physiological roles. The uracil-DNA glycos­
ylase (and most likely other DNA glycosylases as well)
employs the strategy first defined for a bacterial DNA-
Key words: DNA polymerase, repair, ligase
DNASE IV (FEN-1, MF1, S. POMBE Rad2
HOMOLOGUE)
E. coli DNA polymerase I, and related bacterial enzymes such
74
T. Lindahl
5' — P —p P —i” P “T"
G
C
U C
G G
p”
T " P "7~ P“
7 “3'
G
C
A
T
3'—J— P —!— P —1— P —
T
A
P —f—p —L_ p — 5'
Uracil-DNA
glycosylase
S' — p —]— P —j— P —p P —p P —p
G
C
G
A
C
G
G
C
T
P —p
3'
T
A
3'-J- P —I— P —L- P —L- P —L- P —L- p — 5'
AP endonuclease (APE, HAP1)
T
p\
°H X
5'— P —fP —p P —p P —p P —p
G
C
C
G
G
3' —I— P —L
G
C
3'
A
T
T
A
P —L- p —i- p —L- P —L- P — 5'
x>
X
O Xi
DNA polymerase [i
DNA polymerase 8 or e
(and accessory factors)
o,
o° y
5' — p
|
G
C
p
P
{ p —p
C
G
C
G
p —p p —p p - r
G
A
T
C
T
A
3'
X /O
0
x
*7 /O
5'— p— p p p p p — p p ~ r p ~~r°HV " 3'
G
C
C
G
R
T
C
G
G
C
T
A
3‘ —1— P—L- P —* - P —L- P —L- P —L- P — 5'
3' —I— P —*— P —*- P —*- P —L- P —L- P — 5'
5' -> 3‘ exonuclease
(DNase IV, MF1, FEN-1)
OH
5 ‘ — P —p
G
C
P —( P “ p
C
C
G
G
P —p P —p P H G A
T
C T
A
3'
S' — P —p
P —p P “ p P —p P —( P —p 3'
G
C
C
6
fl
T
C
G
G
C
T
A
3 -_ i~ p —L. p —1— p —L_ p —L- p - i - p — 5'
3'—I— P —L- P —L- p —1- P —L- P —1— P — 5'
DNA ligase HI / XRCC1
5 '— P —p P —p P —p P —p P ~ t- P —p 3'
G
C
C
G
A
T
C
G
G
C
T
A
3 '—I— P - Í - P - l - P —L- P - i - P - 1- P— 5'
DNA ligase I
5 '“
P —p P—p P —p P —p P —p P —p
3'
G
CC
G
fl
T
C
GG
C
T
A
3 ‘—I— P - 1- P - 1- P —* - P —* - P —* - P — 5'
as Taq polymerase, contain a distinct N-terminal domain with
5'-nuclease activity. This is the domain removed from E. coli
Poll on generation of the active Klenow fragment. A main
function of the N-terminal domain is to participate in the
removal of 5'-terminal primer RNA sequences from Okazaki
fragments during lagging-strand DNA replication. DNA poly­
merases in mammalian cells do not contain a separate domain
with 5' nuclease activity. However, a distinct 43 kDa
mammalian nuclear enzyme with catalytic properties very
similar to the 5 ' nuclease function of E. coli Poll but not cova­
lently bound to a DNA polymerase was discovered many years
ago and called DNase IV. Recent studies on this enzyme have
demonstrated significant sequence homology with the Nterminal domain of E. coli Poll (Fig. 2), further strengthening
the similarity between these two functions (Robins et al.,
1994). Thus, amino acid residues conserved within the 5'
nuclease domain of microbial DNA polymerases are also
largely retained in human DNase IV. Recently, a related gene
encoding a 5' nuclease, distinct from Poll, was also detected
Fig. 1. Branched pathway of DNA
base-excision repair, resulting in
heterogeneity of repair patch sizes.
The human enzymes that tentatively
account for each separate step are
indicated. From Lindahl et al. (1995).
in E. coli (Sayers, 1994). Using human cell-free systems for
DNA replication, several groups have shown that DNase IV is
required for complete removal of RNA primers from Okazaki
fragments, and the alternative name ‘maturation factor 1’
(MF1) has been used to describe this activity (Ishimi et al.,
1988; Goulian et al., 1990; W aga et al., 1994). A striking
property of the 5' nuclease function of E. coli Poll (Lyamichev
et al., 1993) is its ability to act as a structure-specific endonu­
clease to remove an overhang, or flap, structure of a displaced
single strand of the parental DNA molecule by cleavage at the
branch point (right side of Fig. 1). Such an activity has also
been found in mammalian cell extracts and termed flap
endonuclease (FEN-1; Harrington and Lieber, 1994, 1995); it
is due to the 43 kDa DNase IV. Finally, a cDNA encoding the
human homologue of the Schizosaccharomyces pombe rad2
gene product has been cloned and encodes DNase IV. The
phenotype of S. pombe rad.2 mutants is interesting: mutants are
viable but hypersensitive to ultraviolet light, and show a high
rate of spontaneous chromosome loss. Moreover, cellular over-
Recognition and processing of damaged DNA
Ec
Hu
75
1 MVOIPONPLILVDGSSYLYRAYHAFPP ■ ■ ■ .LTNSAGEPTGAMYGVLNML 46
=1
: :|:|
29
I
Il
II h = h
RKVAIDASMSIYOFLIAVROGGDVT.ONEF.GETTSHLMGMF. .Y 69
47 RSLIMOYKPTHA A W F DAKGKTFRDELFEHYKSHRPPMPDDLRAOI....
I : I
:
I I I: I
= =1 I
=
=
92
I
70 RTIKMMENGIKPVYVFDGKPPOLKSGELAK ■RSERRAEAEKOLOOAOAAGA 119
93 ...................EPLHAMVKAMGLPLLAVSGVEADDVIGTLAR 123
:
:
::
||: |
|
:
||:
:
|
120 EOEVEKFTKRLVKVTKOHNDECKHLLSLMGIPYLDAPS .EAEASCAALV. 167
124 EAEKAGRPVLISTGDKDMAOLVTPNI TL INTMTNTI....... LGPEEV
III:
:| I I
:I
h
=1
165
168 . . .KAGKVYAAATEDMDCLTFGSP ..VTiMRHT.TASEAKKLPIOEFHLSRI 212
166 VNKYGVPPHT,TTDFIiATjMGDfíSDNIPGVPGVGEKTAOALLOGLGGLDTLY 215
:
I:
I :I
hl
II
== hl I I hi
=== =
213 LOELGLNOEOFVDLCILLG..fiDYCESIRGIQPKRAVDLIOKHKSIEEIV 260
ACTIVE
SITE
NH2
....
1..... *
Fig. 2. Alignment o f the 5'—>3' exonuclease
domain o f E. coli DNA polymerase I (Ec) with
human DNase IV (Hu). The first 215 residues of
DNA polymerase I contain the sites conserved
among 10 prokaryotic exonucleases; these are
underlined. Residues identical in all 10
exonucleases are in bold type. This domain was
compared to the part o f DNase IV that is
conserved in the eukaryotic Rad2 family, where
residue 29 is the beginning o f the first conserved
segment. Underlined in the human sequence are
the two regions of the protein that are most
conserved with other members of the Rad2
family. Identical residues are connected by
vertical bars, and double dots show similar
residues. From Robins et al. (1994).
CONSERVED
PEPTIDE
1
COOH
DNA LIGASE I
Fig. 3. Schematic representation
o f DNA ligase I, III and IV
aligned at their active sites. The
active site m otif and the
conserved peptide found in all
ACTIVE
CONSERVED
SITE
eukaryotic DNA ligases are
PEPTIDE
DNA LIGASE III
indicated by solid bars. The
NH2shaded box represents the C■COOH
terminal catalytic domain of
103 kDa
DNA ligase I. The predicted
100 kDa
by SDS-PAGE
molecular mass of the
polypeptide encoded by each
ACTIVE
CONSERVED
respective open reading frame is
PEPTIDE
SITE
COOH
indicated, while figures to the
NH2DNA LIGASE IV
side in bold typeface give the
apparent molecular mass o f each
96 kDa 100 kDa
protein as estimated by SDSby SDS-PAGE
PAGE; figures in brackets refer
to the catalytic fragment of DNA ligase I. The three full-length enzymes are o f similar size, but DNA ligase I migrates anomalously slowly
during SDS-PAGE due to its hydrophilic N-terminal region. From Wei et al. (1995).
■ 102 kDa
(78 kDa)
expression of the Rad2 protein leads to inviability associated
with fragmentation of the cell nucleus (Murray et al., 1994).
The proposed function of the human enzyme in DNA replica­
tion and repair seems in agreement with this phenotype of S.
pombe ra d i mutants.
HUMAN DNA LIGASES
By standard methods of enzyme purification and characteriza­
tion, we found three distinct DNA ligases, I-III, in mammalian
cell nuclei. A cDNA encoding the major enzyme in proliferat­
ing human cells was cloned, and the gene mapped to chromo­
some 19q 13.2-13.3. Data with a DNA ligase I-defective human
cell line implicate this enzyme in joining of Okazaki fragments
during DNA replication (Prigent et al., 1994), in agreement
with its apparent role in SV40 DNA replication in human cell
extracts (Waga et al., 1994). DNA ligase I shows distinct
homology with the Saccharomyces cerevisiae CDC9 and S.
pombe cd cl7 + gene products. All eukaryotic DNA ligases
share a highly conserved stretch of 16 amino acids, located
125 kDa
(85 kDa)
by SDS-PAGE
close to the C terminus of DNA ligase I, which is likely to be
required for generation and/or processing of the DNA-AMP
reaction intermediate. A search of a comprehensive human
cDNA library for expressed sequence tags corresponding to
this region revealed cDNAs encoding two DNA ligases addi­
tional to DNA ligase I, the previously described DNA ligase
III and a novel DNA ligase IV (Wei et al., 1995). DNA ligases
I, III and IV all are -1 0 0 kDa proteins, and their outline struc­
tures are shown in Fig. 3. The minor DNA ligases III and IV
map to human chromosomes 17q 11.2-12 and 13q33-34,
respectively. The 70 kDa DNA ligase II appears identical, or
closely similar, to the C-terminal two-thirds of DNA ligase III;
it has a blocked N-terminal residue indicating that it is a
primary translation product, and is probably derived from the
same gene as DNA ligase III by alternative splicing or by
employing a different translational start (Roberts et al., 1994;
Husain et al., 1995). The tissue distribution of DNA ligases II
and III is quite different, with ligase II high in liver, whereas
ligase III is high in testes and thymus.
The central domains of the different DNA ligases show con­
siderable sequence homology between each other and also with
76
T. Lindahl
RNA capping enzymes, which act by a similar mechanism of
covalent catalysis involving an enzyme-nucleotide intermedi­
ate (Shuman et al., 1994). However, the N-terminal regions of
DNA ligases I and III, and the C-terminal region of DNA ligase
IV, are completely different from each other and from other
proteins in data banks. It seems likely that these regions are
involved in specific protein-protein interactions, conferring
individual specificities on the various DNA ligases in different
replication, repair, and recombination events. Thus, the
hydrophilic N-terminal region of DNA ligase I may bind to
other proteins required for lagging-strand replication. The best
characterized interaction at present is the tight complex formed
between DNA ligase III and the 70 kDa XRCC1 protein
(Caldecott et al., 1994). Rodent cell lines defective in XRCC1
exhibit hypersensitivity to alkylating agents and ionizing
radiation, suggesting a role in the base excision-repair pathway
for the ligase III/XRCC1 complex. The N-terminal region of
DNA ligase III has an unusual zinc finger (Wei et al., 1995)
which seems very similar to the two zinc fingers found in the
N-terminal region of poly(ADP-ribose) polymerase, another
nuclear enzyme that binds specifically to strand interruptions
in DNA (Gradwohl et al., 1990).
SIGNALS AT DNA STRAND BREAKS
In response to a DNA strand interruption generated by ionizing
radiation or an alkylating agent, mammalian cells very rapidly
synthesize poly(ADP-ribose). The abundant enzyme responsi­
ble, poly(ADP-ribose) polymerase (PARP), is associated with
the nuclear matrix. It undergoes extensive, dramatic but
transient automodification in response to DNA damage, with
production of long branched polymers of poly(ADP-ribose)
from NAD. These are rapidly degraded by a specific glycohydrolase after synthesis. Recent investigations with cell-free
systems have shown that poly(ADP-ribose) synthesis is not
directly required for DNA repair, but the exact function of
these long polymers, produced in response to DNA damage,
remains unclear (Satoh and Lindahl, 1992; Smulson et al.,
1994; Satoh et al., 1994). They may serve to reorganize
chromatin structure at a lesion site (Panzeter et al., 1993),
perhaps in order to suppress homologous recombination in
tandem repeat sequences and resulting genomic instability.
‘Double knock-out’ mice lacking PARP are viable and are not
markedly hypersensitive to DNA-damaging agents (Wang et
al., 1995) but little information is currently available on the
properties of cell lines derived from those animals.
In addition to PARP, several other nuclear enzymes compete
for strand breaks in DNA, including the large DNA-dependent
protein kinase defective in scid cells (Blunt et al., 1995). The
binding and activation of such enzymes at DNA strand inter­
ruptions apparently leads to a stress response with generation
of a signal that accounts for accumulation and increased levels
of p53 protein in damaged cells (Lu and Lane, 1993).
Normally, p53 has a short half-life in vivo and is degraded by
a ubiquitin-dependent pathway. Thus, DNA damage and
resultant activation of nuclear enzymes, such as PARP, DNAdependent protein kinase, and other protein kinases, may serve
to interfere with ubiquitin-conjugating enzymes, to allow for
p53 stabilisation and accumulation. It is probably relevant in
this context that S. cerevisiae RAD6 mutants are hypersensi­
tive to DNA damage and also are defective in ubiquitin con­
jugation (Jentsch et al., 1987). The biochemical details of this
stress response in mammalian cells, when known, should
elucidate an important mechanism for arrest of cell division in
response to DNA damage.
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