Genome maintenance mechanisms.pdf

insight review articles

Genome maintenance mechanisms

for preventing cancer

Jan H. J. Hoeijmakers

MGC Department of Cell Biology and Genetics, Centre for Biomedical Genetics, Erasmus University, PO Box 1738, 3000DR Rotterdam,

The Netherlands (e-mail: Hoeijmakers@gen.fgg.eur.nl)

The early notion that cancer is caused by mutations in genes critical for the control of cell growth implied

that genome stability is important for preventing oncogenesis. During the past decade, knowledge about the

mechanisms by which genes erode and the molecular machinery designed to counteract this time-dependent

genetic degeneration has increased markedly. At the same time, it has become apparent that inherited or

acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer. This

review summarizes the main DNA caretaking systems and their impact on genome stability and

carcinogenesis.

C ancer is a disease of our genes. Over time,

miscoding uracil, hypoxanthine, xanthine and thymine,

respectively 4 . Figure 1a summarizes some of the most

common types of DNA damage and their sources.

DNA accumulates changes that activate proto-

oncogenes and inactivate tumour-suppressor

genes. The genetic instability driving

tumorigenesis is fuelled by DNA damage and

by errors made by the DNA machinery. However,

‘spontaneous’ mutations are insufficient to explain the

lifetime cancer risk 1 . Indeed, numerous links have been

identified between oncogenesis and acquired or inherited

faulty genome guardians that cause a ‘mutator’

phenotype, highlighting the key role of DNA protection

systems in tumour prevention. Here I focus on the main

DNA maintenance mechanisms operating in mammals —

nucleotide- and base-excision repair, homologous

recombination, end joining, mismatch repair and

telomere metabolism — and their relevance for cancer.

The consequences of DNA injury

The outcome of DNA damage is diverse and generally

adverse (Fig. 1b). Acute effects arise from disturbed DNA

metabolism, triggering cell-cycle arrest or cell death. Long-

term effects result from irreversible mutations contributing

to oncogenesis.

Many lesions block transcription, which in effect inacti-

vates every gene containing damage on the transcribed

strand — an outcome directly related to gene length. This

has elicited the development of a dedicated repair system,

transcription-coupled repair (TCR), which displaces or

removes the stalled RNA polymerase and assures high-

priority repair. Transcriptional stress, arising from

persistent blockage of RNA synthesis, constitutes an effi-

cient trigger for p53-dependent apoptosis (see ref. 5 and the

article in this issue by Evan and Vousden, pages 342–348),

which may be a significant anti-cancer mechanism.

Lesions also interfere with DNA replication. Recently, a

growing class of DNA polymerases, numbered z to k , was

discovered which seems devoted specifically to overcoming

damage-induced replicational stress 6,7 . These special

polymerases take over temporarily from the blocked

replicative DNA polymerase-

A plethora of damages in DNA

The physicochemical constitution of our genes does not

guarantee life-long stability or proper function. A perplex-

ing diversity of lesions arises in DNA from three main

causes. First, environmental agents such as the ultraviolet

(UV) component of sunlight, ionizing radiation and

numerous genotoxic chemicals cause alterations in DNA

structure, which, if left unrepaired, may lead to mutations

that enhance cancer risk. A pronounced example is

exposure to genotoxic compounds in cigarette smoke,

which are responsible for the most frequent cancer in

Western men. Second, (by)products of normal cellular

metabolism constitute a permanent enemy to DNA

integrity from within. These include reactive oxygen species

(superoxide anions, hydroxyl radicals and hydrogen

peroxide) derived from oxidative respiration and products

of lipid peroxidation. Over 100 oxidative modifications

have been identified in DNA 2 . Evolution has invested

significantly in reducing the price of its own metabolism by

implementing an intricate antioxidant defence system

composed of enzymatic (superoxide dismutase, catalase,

glutathione peroxidase and peroxyredoxins) and low-

molecular-mass scavengers (such as glutathione) 3 . Finally,

some chemical bonds in DNA tend to spontaneously

disintegrate under physiological conditions. Hydrolysis of

nucleotide residues leaves non-instructive abasic sites.

Spontaneous or induced deamination of cytosine, adenine,

guanine or 5-methylcytosine converts these bases to the

;

(pol

;

), and possibly

from pol

(Fig. 2, follow upper strand). They have more

flexible base-pairing properties permitting translesion

synthesis, with each polymerase probably designed for a

specific category of injury. The number of polymerases

preferring damaged templates currently exceeds that for

undamaged DNA, which illustrates the magnitude of the

problem. But this solution generally comes at the expense of

a higher error rate. In fact, this process is responsible for

most of damage-induced point mutations 8 and is thus

particularly relevant for oncogenesis. Nevertheless, transle-

sion polymerases still protect the genome. For instance,

inherited defects in pol-

, which specializes in relatively

error-free bypassing of UV-induced cyclobutane pyrimi-

dine dimers, cause the variant form of the skin cancer-

prone disorder xeroderma pigmentosum 9,10 . In the yeast

Saccharomyces cerevisiae , a second, probably even more

important pathway exists that allows error-free bypass of

lesions 8 . This mechanism is based on reinitiation of DNA

NATURE | VOL 411 | 17 MAY 2001 | www.nature.com

366

insight review articles

Damaging agent

Consequences

X-rays

Oxygen radicals

Alkylating agents

Spontaneous reactions

(Transient)

cell-cycle

arrest

X-rays

Anti-tumour agents

( cis- Pt, MMC)

UV light

Polycyclic aromatic

hydrocarbons

Replication

errors

U G

G G

Inhibition of:

• Transcription

• Replication

• Chromosome

segregation

Apoptosis

(cell death)

Uracil

Abasic site

8-Oxoguanine

Single-strand break

(6–4)PP

Bulky adduct

CPD

Interstrand cross-link

Double-strand break

A–G Mismatch

T–C Mismatch

Insertion

Deletion

Mutations

Chromosome

aberrations

Base-excision

repair (BER)

Nucleotide-excision

repair (NER)

Recombinational

repair (HR, EJ)

Mismatch repair

Cancer

Ageing

Inborn

disease

Repair process

Figure 1 DNA damage, repair mechanisms and consequences. a , Common DNA damaging agents (top); examples of DNA lesions induced by these agents (middle); and most

relevant DNA repair mechanism responsible for the removal of the lesions (bottom). b , Acute effects of DNA damage on cell-cycle progression, leading to transient arrest in the G1,

S, G2 and M phases (top), and on DNA metabolism (middle). Long-term consequences of DNA injury (bottom) include permanent changes in the DNA sequence (point mutations

affecting single genes or chromosome aberrations which may involve multiple genes) and their biological effects. Abbreviations: cis -Pt and MMC, cisplatin and mitomycin C,

respectively (both DNA-crosslinking agents); (6–4)PP and CPD, 6–4 photoproduct and cyclobutane pyrimidine dimer, respectively (both induced by UV light); BER and NER, base-

and nucleotide-excision repair, respectively; HR, homologous recombination; EJ, end joining.

replication downstream of the blocking injury. The resulting gap is

filled in by recombinational replication, using the newly synthesized

complementary strand as a template and ignoring the original

lesion-containing one (Fig. 2, follow lower strand). Yeast proteins

implicated in this process, such as the Ubc13/Mms2 complex, are

conserved all the way to mammals. Thus, this largely unexplored

system undoubtedly exists in humans and may be important in

carcinogenesis. The endpoint of both of these pathways is that dam-

age persists and — when unrepaired — will cause similar problems in

subsequent rounds of replication. This is particularly relevant for

damage that is not efficiently recognized by any mammalian repair

process, such as cyclobutane pyrimidine dimers.

Double-strand DNA breaks (DSBs) induced by X-rays, chemicals

or during replication of single-strand breaks (SSBs) and presumably

during repair of interstrand crosslinks are particularly relevant for

the recombination machinery. Cells with specialized DNA recombi-

nation activities, such as B- and T-cells, may be very sensitive to DSBs

when they are rearranging their immunoglobin or T-cell-receptor

genes. This explains the frequent involvement of these genetic loci in

oncogenic translocations in leukaemia and lymphomas and the

preferential induction of these cancers by ionizing irradiation. DSBs

also pose problems during mitosis, as intact chromosomes are a

prerequisite for proper chromosome segregation during cell

division. Thus, these lesions frequently induce various sorts of

chromosomal aberrations, including aneuploidy, deletions (loss of

heterozygosity) and chromosomal translocations — events which

are all intimately associated with carcinogenesis.

The cell-cycle machinery somehow senses genome injury and

arrests at specific checkpoints in G1, S, G2 and M to allow repair of

lesions before they are converted into permanent mutations

(reviewed in ref. 11). Lesion detection may occur by blocked

transcription, replication or specialized sensors. When damage is too

significant, a cell may opt for the ultimate mode of rescue by initiating

apoptosis at the expense of a whole cell (see review by Evan and

Vousden, pages 342–348).

DNA damage repair systems

In view of the plethora of types of lesions, no single repair process can

cope with all kinds of damage. Instead, evolution has moulded a

tapestry of sophisticated, interwoven DNA repair systems that as a

whole cover most (but not all) of the insults inflicted on a cell’s vital

genetic information. Inherited defects in any of these pathways in

general predisposes to malignancy (Table 1). Because the problem of

DNA damage has existed ab initio , DNA repair systems must have

arisen early in evolution. This explains why all known repair

pathways are highly conserved (usually across the pro/eukaryotic

evolutionary border). At least four main, partly overlapping damage

repair pathways operate in mammals — nucleotide-excision repair

(NER), base-excision repair (BER), homologous recombination and

end joining 12,13 . The division of tasks between them can be roughly

defined as follows (see also Fig. 1a).

NER deals with the wide class of helix-distorting lesions that

interfere with base pairing and generally obstruct transcription and

normal replication. Small chemical alterations of bases are targeted

by BER. These lesions may or may not impede transcription and

replication, although they frequently miscode. BER is therefore par-

ticularly relevant for preventing mutagenesis. Most NER lesions arise

from exogenous sources (except for some oxidative lesions), whereas

BER is mostly, but not exclusively, concerned with damage of

endogenous origin. Lesions for these two repair processes affect only

NATURE | VOL 411 | 17 MAY 2001 | www.nature.com

367

insight review articles

Table 1 Human syndromes with defective genome maintenance

Syndrome

Affected

Main type

Major cancer

Replication-blocking

lesion

maintenance

of genome

predisposition

mechanism

instability

DNA pol d/e

Xeroderma

NER (

TCR)

Point mutations

UV-induced

pigmentosum

skin cancer

Cockayne syndrome

TCR

Point mutations

None*

Trichothiodystrophy

NER / TCR

Point mutations

None*

Ataxia

DSB response/repair

Chromosome

Lymphomas

telangiectasia (AT)

aberrations

Polymerase switch

Translesion synthesis by

specialized DNA polymerase ( z – k )

(frequently error-prone)

AT-like disorder

DSB response/repair

Chromosome

Lymphomas

DNA pol

aberrations

Nijmegen breakage

DSB response/repair

Chromosome

Lymphomas

syndrome

aberrations

BRCA 1/BRCA2

Chromosome

Breast (ovarian)

aberrations

cancer

Werner syndrome

HR?/TLS?

Chromosome

Various cancers

aberrations

Bloom syndrome

HR?

Chromosome

Leukaemia,

DNA pol

–

aberrations

lymphoma,

(SCE

)

others

Rothmund–Thomson HR?

Chromosome

Osteosarcoma

syndrome

aberrations

Ligase IV deficiency†

Recombination

Leukaemia(?)

fidelity

Template switch

Recombination-dependent

daughter-strand gap repair

(principally error-free)

HNPCC

MMR

Point mutations

Colorectal cancer

Xeroderma

TLS‡

Point mutations

UV-induced

pigmentosum

skin cancer

variant

*Defect in transcription-coupled repair triggers apoptosis, which may protect against UV-

induced cancer.

†One patient with leukaemia and radiosensitivity described with active-site mutation in ligase IV.

‡Specific defect in relatively error-free bypass replication of UV-induced cyclobutane pyrimidine

dimers.

Abbreviations: BER, base-excision repair; DSB, double-strand break; HNPCC, hereditary non-

polyposis colorectal cancer; HR, homologous recombination; MMR, mismatch repair; NER,

nucleotide-excision repair; SCE, sister-chromatid exchange; TCR, transcription-coupled repair;

TLS, translesion synthesis.

the DSB problem. Homologous recombination seems to dominate in

S and G2 when the DNA is replicated, providing a pristine second

copy of the sequence (sister chromatid) for aligning the breaks. In

contrast, the less-accurate end joining is most relevant in the G1

phase of the cell cycle, when a second copy is not available 14 .

Finally, some single repair proteins directly revert certain injuries,

such as O 6 -methylguanine methyltransferase, which removes

O 6 -methyl guanine. This highly mutagenic lesion permits base

pairing with both C or T and is capable of fooling the mismatch repair

system into triggering futile rounds of mismatch removal and subse-

quent reincorporation of the erroneous base by repair replication.

The dedicated methyl transferase specifically removes the non-native

methyl group from the guanine residue and transfers it to an internal

cysteine. However, in doing so, the protein irreversibly inactivates

itself 13 . This illustrates how in some situations an entire protein may

be sacrificed for the repair of a single damaged base. Below I describe

the four main multi-step damage repair processes in mammals and

their relevance for preventing cancer.

Figure 2 Mechanisms of replicational bypass of DNA lesions. Lesions in the DNA

template (indicated by an ‘X’) may be bypassed by the replication apparatus in two

different ways: DNA polymerase switch (upper strand) and template switch (lower

strand). In the DNA polymerase switch, the regular DNA polymerase (in this case

pol

, carrying out leading-strand synthesis) is arrested at the site of the damage.

A specific translesion polymerase (polz–k), or a combination of these polymerases,

takes over synthesis to bypass the injured site, after which the regular polymerase

continues. This process can be highly error-prone. In the template switch (model), the

regular DNA polymerase (in this case pol

;

, responsible for lagging-strand synthesis)

is arrested at a damaged site. The resulting gap in the newly synthesized strand is

filled in using the undamaged, newly synthesized leading strand via recombinational

strand exchange (or alternatively by fork regression and annealing of the new strand,

not shown). This mechanism may involve specific factors as well as members of the

RAD52 family implicated in homologous recombination repair. In principle, this mode

of lesion bypass is error-free. Note that in both of these processes the lesion remains

and that the two scenarios may apply to both strands.

Nucleotide-excision repair and transcription-coupled repair

Of all repair systems, NER is the most versatile in terms of lesion

recognition. Two NER subpathways exist with partly distinct sub-

strate specificity: global genome NER (GG-NER) surveys the entire

genome for distorting injury, and transcription-coupled repair

(TCR) focuses on damage that blocks elongating RNA

polymerases 15 . Box 1 presents the most likely mechanisms of action

for these pathways (and see refs 16, 17).

one of the DNA strands. In a ‘cut-and-patch’-type reaction, the

injury (with or without some flanking sequences) is taken out and the

resulting single-stranded gap is filled in using the intact complemen-

tary strand as template.

DSBs are more problematic, as both strands are affected. To

properly heal such breaks the cell has to know which ends belong

together, a difficult task given the size of the mammalian genome.

Two pathways, homologous recombination and end joining (and

presumably additional back-up systems), were developed for solving

NER, TCR and cancer

At least three syndromes are associated with inborn defects in NER

(Table 1): xeroderma pigmentosum, Cockayne syndrome and

trichothiodystrophy (TTD), all characterized by exquisite sun sensi-

tivity 18,19 . The prototype repair disorder, xeroderma pigmentosum,

NATURE | VOL 411 | 17 MAY 2001 | www.nature.com

368

insight review articles

Box 1

Model for mechanism of global genome nucleotide-excision repair and transcription-coupled repair

The GG-NER-specific complex XPC-hHR23B screens first on

the basis of disrupted base pairing 53 , instead of lesions per se.

This explains why mildly distorting injury such as cyclobutane

pyrimidine dimers are poorly repaired 54 . In TCR, the ability of a

lesion (whether of the NER- or BER-type) to block RNA

polymerase seems critical (stage I in the figure opposite). The

stalled polymerase must be displaced to make the injury

accessible for repair 55 , and this requires at least two

TCR-specific factors: CSB and CSA. The subsequent stages

of GG-NER and TCR may be identical. The XPB and XPD

helicases of the multi-subunit transcription factor TFIIH open

~30 base pairs of DNA around the damage (II). XPA probably

confirms the presence of damage by probing for abnormal

backbone structure 56 , and when absent aborts NER 53 . The

single-stranded-binding protein RPA (replication protein A)

stabilizes the open intermediate by binding to the undamaged

strand (III). The use of subsequent factors, each with limited

capacity for lesion detection in toto , still allows very high

damage specificity 57 . The endonuclease duo of the NER team,

XPG and ERCC1/XPF, respectively cleave 38 and 58 of the

borders of the opened stretch only in the damaged strand,

generating a 24–32-base oligonucleotide containing the

injury (IV). The regular DNA replication machinery then

completes the repair by filling the gap (V). In total, 25 or more

proteins participate in NER. In vivo studies indicate that the

NER machinery is assembled in a step-wise fashion from

individual components at the site of a lesion. After a single repair

event (which takes several minutes) the entire complex is

disassembled again 58 .

Transcription-coupled repair

Global genome NER

NER lesions

(e.g. due to UV damage)

Elongating Pol II-blocking lesions

(e.g. due to UV and oxidative damage)

Genome overall

Transcribed DNA

Elongating

RNA Pol II

CSB

XPC-hHR23B

Pol II

CSA

TFIIH

XPG

others

TFIIH

XPG

TFIIH

XPA

RPA

TFIIH

III

RPA

ERCC1-XPF

TFIIH

RPA

Replication

factors

exhibits a dramatic >1000-fold incidence of sun-induced skin

cancer. Frequency of internal tumours is modestly elevated and

accelerated neurodegeneration is often noted. The disorder arises

from mutations in one of seven genes ( XPA – XPG ). Cockayne

syndrome, caused by mutation in the CSA or CSB genes, is a

TCR-specific disorder that is remarkably dissimilar from xeroderma

pigmentosum. No predisposition to cancer is observed, which may

be explained by the fact that the TCR defect causes Cockayne

syndrome cells to be particularly sensitive to lesion-induced

apoptosis, thereby protecting against tumorigenesis. Physical and

neurological development are impaired, resulting in dwarphism and

dysmyelination. The syndrome includes features of premature age-

ing, which may be related to the increased trigger for apoptosis

induced by transcriptional arrest from endogenous lesions in

combination with the TCR defect . TTD is a condition sharing many

symptoms with Cockayne syndrome, but with the additional

hallmarks of brittle hair, nails and scaly skin. Mutations in the XPD or

XPB genes can give rise to all three diseases. This puzzle is explained

by the fact that, as subunits of TFIIH, XPB and XPD have dual

functions: NER and transcription initiation. Mutations may not only

compromise NER, but also affect transcription, causing develop-

mental delay and reduced expression of the matrix proteins that

causes brittle hair and scaly skin 20 .

For almost all NER factors, mouse mutants have been generated 21 .

Overall, the NER defect is accurately preserved, although cancer

predisposition is more pronounced and neurological complications

are milder in mice. Moreover, mice exhibit features of premature

ageing.

NATURE | VOL 411 | 17 MAY 2001 | www.nature.com

369

insight review articles

Box 2

Mechanism for base-excision repair

A battery of glycosylases, each dealing

with a relatively narrow, partially

overlapping spectrum of lesions, feeds

into a core reaction. Glycosylases flip

the suspected base out of the helix by

DNA backbone compression to

accommodate it in an internal cavity of

the protein. Inside the protein, the

damaged base is cleaved from the

sugar-phosphate backbone (stage I in

the figure). The resulting abasic site can

also occur spontaneously by

hydrolysis. The core BER reaction is

initiated by strand incision at the abasic

site by the APE1 endonuclease (II).

Poly(ADP-ribose) polymerase (PARP),

which binds to and is activated by DNA

strand breaks, and the recently

identified polynucleotide kinase (PNK) 59

may be important when BER is initiated

from a SSB to protect and trim the

ends for repair synthesis (III). In

mammals, the so-called short-patch

repair is the dominant mode for the

remainder of the reaction. DNA pol

Reactive oxygen species

Methylation, deamination

X rays

(single-stranded break)

AACCGT

ACT GGC

AACCGT X

A CT GGC

T T GGCA

T GA CCG

T T GGC

T GACCG

Spontaneous hydrolysis

(abasic site)

DNA

glycosylase

XRCC1

PARP

AACCGT

ACT GGC

AACCGT

ACT GGC

T T GGC

T GACCG

T T GGCA

T GA CCG

APE1

PNK

OH P

AACC

AAC

CGT

A CT GGC

III

T T GGCA

T GA CCG

DNA pol b

XRCC1

PCNA

DNA pol d/e

+dNTPs

+dGTP

AACCGT

GGC

VII

TTGGC

TGACCG

performs a one-nucleotide gap-filling

reaction (IV) and removes the

58-terminal baseless sugar residue via

its lyase activity (V); this is then followed

by sealing of the remaining nick by the

XRCC1–ligase3 complex (VI). The

XRCC1 scaffold protein interacts with

most of the above BER core

components and may therefore be

instrumental in protein exchange. The

long-patch repair mode involves DNA

pol

FEN1

TGACCG

AACCGT

VIII

TTGGC

DNA

ligase1

DNA

ligase 3

ACTG

AACCGT

ACT GGC

AACCGT

T T GGCAC

TGACC

T T GGCA

T GA CCG

Long-patch BER

(Minor pathway)

Short-patch BER

(Main pathway)

and proliferating cell

nuclear antigen (PCNA) for repair synthesis (2–10 bases) as well as the FEN1 endonuclease to remove the displaced DNA flap and DNA ligase 1

for sealing (VII–IX). The above BER reaction operates across the genome. However, some BER lesions block transcription, and in this case the

problem is dealt with by the TCR pathway described above, including TFIIH, XPG (which also stimulates some of the glycosylases) and probably

the remainder of the core NER apparatus.

, pol

;

Base-excision repair

BER is the main guardian against damage due to cellular metabolism,

including that resulting from reactive oxygen species, methylation,

deamination and hydroxylation. The molecular mechanism 13 has

been resolved to the tertiary structure of all core components 22–24 and

is explained in Box 2.

the glycosylases that cannot be further processed 13,25 . Interestingly,

specific polymorphisms in XRCC1 seem associated with lung and

other cancers 26 .

DSB repair: homologous recombination and end joining

DSBs arise from ionizing radiation or X-rays, free radicals, chemicals

and during replication of a SSB. After DSB detection, a complex

cascade of reactions is triggered aimed at halting the cell-cycle

machinery and recruiting repair factors 11,27 (Fig. 5). One of the early

initiators is the ataxia telangiectasia mutated (ATM) protein kinase,

which is defective in the cancer-prone, X-ray-sensitive syndrome

ataxia telangiectasia 28 . Arrest in G1 is mediated via p53. Another early

event, which depends on the giant protein-kinases ATM, ATR (ataxia

telangiectasia related) and DNA-PK cs , is phosphorylation of histone

H2AX in the DNA domain next to the DSB over a megadalton

distance 29 . This may provide a local chromatin state required for the

complex repair reactions or for recruiting repair proteins. Homolo-

gous recombination and end joining are the main repair modes.

When, after replication, a second identical DNA copy is available,

homologous recombination seems to be preferred; otherwise cells

BER and cancer

No human disorders caused by inherited BER deficiencies have been

identified. Mouse models generated in recent years may provide an

explanation: knockout of individual glycosylases does not cause an

overt phenotype, which is explained by partial redundancy between

different glycosylases 13,25 and overlap with TCR. In fact, even a

number of double mutants show only mild phenotypes, although

mutagenesis and cancer susceptibility are probably increased. But

inactivation of BER core proteins induces embryonic lethality,

highlighting the vital importance of the process as a whole. This

might be due to the contribution of spontaneously occurring

abasic sites and SSBs that directly feed into the BER core reaction

(Box 2) and/or to the generation of reaction intermediates by

NATURE | VOL 411 | 17 MAY 2001 | www.nature.com

370

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: