Jan H.J. Hoeijmakers

Genome maintenance mechanisms for preventing cancer.

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.

DNA Damage, Aging, and Cancer

NA damage has emerged as a major culprit in cancer and many diseases related to aging. The stability of the genome is supported by an intricate machinery of repair, damage tolerance, and checkpoint pathways that counteracts DNA damage. In addition, DNA damage and other stresses can trigger a highly conserved, anticancer, antiaging survival response that suppresses metabolism and growth and boosts defenses that maintain the integrity of the cell. Induction of the survival response may allow interventions that improve health and extend the life span. Recently, the first candidate for such interventions, rapamycin (also known as sirolimus), has been identified. 1 Compromised repair systems in tumors also offer opportunities for intervention, making it possible to attack malignant cells in which maintenance of the genome has been weakened. Time-dependent accumulation of damage in cells and organs is associated with gradual functional decline and aging. 2 The molecular basis of this phenomenon is unclear, 3-5 whereas in cancer, DNA alterations are the major culprit. In this review, I present evidence that cancer and diseases of aging are two sides of the DNAdamage problem. An examination of the importance of DNA damage and the systems of genome maintenance in relation to aging is followed by an account of the derailment of genome guardian mechanisms in cancer and of how this cancerspecific phenomenon can be exploited for treatment.

Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms

Many biochemical, physiological and behavioural processes show circadian rhythms which are generated by an internal time-keeping mechanism referred to as the biological clock. According to rapidly developing models, the core oscillator driving this clockis composed of an autoregulatory transcription–(post) translation-based feedback loop involving a set of ‘clock’ genes,. Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cry1 and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Here we show that mice lacking the Cry1 or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity.

Chromosomal stability and the DNA double-stranded break connection

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-stranded breaks in DNA are important threats to genome integrity because they can result in chromosomal aberrations that can affect, simultaneously, many genes, and lead to cell malfunctioning and cell death. These detrimental consequences are counteracted by two mechanistically distinct pathways of double-stranded break repair: homologous recombination and non-homologous end-joining. Recently, unexpected links between these double-stranded break-repair systems, and several human genome instability and cancer predisposition syndromes, have emerged. Now, interactions between both double-stranded break-repair pathways and other cellular processes, such as cell-cycle regulation and replication, are being unveiled.

Xeroderma Pigmentosum Group C Protein Complex Is the Initiator of Global Genome Nucleotide Excision Repair

The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled nucleotide excision repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.

Sequential assembly of the nucleotide excision repair factors in vivo

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes

Cells from patients with the UV-sensitive nucleotide excision repair disorder Cockaynes syndrome (CS) have a specific defect in preferential repair of lesions from the transcribed strand of active genes. This system permits quick resumption of transcription after UV exposure. Here we report the characterization of ERCC6, a gene involved in preferential repair in eukaryotes. ERCC6 corrects the repair defect of CS complementation group B (CS-B). It encodes a protein of 1493 amino acids, containing seven consecutive domains conserved between DNA and RNA helicases. The entire helicase region bears striking homology to segments in recently discovered proteins involved in transcription regulation, chromosome stability, and DNA repair. Mutation analysis of a CS-B patient indicates that the gene is not essential for cell viability and is specific for preferential repair of transcribed sequences.

P53 modulation of TFIIH-associated nucleotide excision repair activity

p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH–associated factors, including transcription–repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand–specific DNA repair, via its C–terminal domain. We also found that wild–type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV–induced dimers is slower in Li–Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

Molecular mechanisms of DNA double-strand break repair

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.

A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis

XPF–ERCC1 endonuclease is required for repair of helix-distorting DNA lesions and cytotoxic DNA interstrand crosslinks. Mild mutations in XPF cause the cancer-prone syndrome xeroderma pigmentosum. A patient presented with a severe XPF mutation leading to profound crosslink sensitivity and dramatic progeroid symptoms. It is not known how unrepaired DNA damage accelerates ageing or its relevance to natural ageing. Here we show a highly significant correlation between the liver transcriptome of old mice and a mouse model of this progeroid syndrome. Expression data from XPF–ERCC1-deficient mice indicate increased cell death and anti-oxidant defences, a shift towards anabolism and reduced growth hormone/insulin-like growth factor 1 (IGF1) signalling, a known regulator of lifespan. Similar changes are seen in wild-type mice in response to chronic genotoxic stress, caloric restriction, or with ageing. We conclude that unrepaired cytotoxic DNA damage induces a highly conserved metabolic response mediated by the IGF1/insulin pathway, which re-allocates resources from growth to somatic preservation and life extension. This highlights a causal contribution of DNA damage to ageing and demonstrates that ageing and end-of-life fitness are determined both by stochastic damage, which is the cause of functional decline, and genetics, which determines the rates of damage accumulation and decline.