Integrating DNA damage repair with the cell cycle

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DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances.

Introduction

During the past few decades the DNA repair field moved from considering repair simply as a variety of enzymatic activities and pathways towards understanding of how the relevant DNA transactions act in the context of the cellular environment and the cell division cycle. In simple terms the cell cycle is driven by cyclin dependent kinases (CDKs) that form a negative feedback loop oscillator [1]. The negative feedback is defined by the fact that high CDK activity drives CDK destruction. Mitotic CDKs have two core subunits, a cyclin that is periodically degraded and a kinase that is regulated by phosphorylation. When cells are born into G1, CDK levels are very low because the Anaphase Promoting Complex (APC/C; a specialised ubiquitin ligase) has been activated by the high CDK required for mitosis and has destroyed the mitotic cyclins. APC/C remains active in G1 and the consequent low CDK activity essentially defines G1.

Mammalian cells enter from G1 into S phase in response to the expression of D-type cyclins induced by growth factor signalling pathways. This causes overall CDK levels to increase and consequently the APC/C is inhibited. During S phase, mitotic cyclins are transcribed and translated and CDK activity thus continues to increase [2], although CDK activity is attenuated by phosphorylation of the kinase subunit by WEE1-related enzymes. As S phase reaches completion, CDK levels continue to increase and cells transit into G2. Although there is not a defined point where S phase ends and G2 starts, it has historically been considered that the completion of bulk DNA synthesis, as measured by flow cytometry, marks the beginning of G2. More recently, it has become apparent that late replicating regions, including fragile sites, are still being replicated during ‘G2’ in many cells [3].

The decision of when to enter mitosis depends on the continued increase in cyclin levels, the attenuation of WEE1-like kinase activities by CDK and an increase in CdDC25 phosphatase activity (that counteracts WEE1-dependent CDK phosphorylation). These events form positive feedback loops that intersect with the negative feedback mechanism to provide bi-stability to the oscillator. The balance of positive and negative activities, and thus the precise timing of mitosis, are modulated by regulatory mechanisms during which CDK interfaces with other mitotic [4] and checkpoint [5••] kinases. Once the bi-stable switch has been thrown [6], cells can be considered to be committed to mitosis. Because high CDK levels activate the APC/C (the negative feedback loop), high CDK activity programs its own destruction by APC/C-dependent cyclin degradation following the metaphase/anaphase transition. Thus, a new cycle begins [7].

It was first shown in fission yeast that CDK activity was required for efficient double strand break (DSB) repair [8]. Mutants impaired for cyclin B, the sole mitotic cyclin in fission yeast, were observed to be sensitive to DNA damage and unable to appropriately accumulate RPA foci in response to DSBs (see Figure 1). Subsequent work in budding yeast demonstrated that CDK phosphorylation directly regulated resection initiators [9, 10] and other enzymatic activities that promote DSB resection [11, 12•]. Similar phenomena were subsequently shown to be conserved in human cells [13, 14, 15, 16, 17, 18].

Section snippets

DSB repair pathway choice: G1 v G2

DSBs are the most toxic DNA lesions [19]. In a haploid organism, a single unrepaired or misrepaired DSB is generally lethal. In diploid organisms, a single DSB may not be cell lethal, but if unrepaired or misrepaired can result in loss of heterozygosity (LOH) and/or generate gross chromosomal rearrangements (GCRs). There are two main pathways to repair DSBs (Figure 1), homologous recombination (HR), which is generally error-free, and Non Homologous End Joining (NHEJ) that frequently introduces

DNA repair in S phase

Some aspects of DNA repair in S phase are specific by definition. For example, lesions such as inter-strand crosslinks (ICLs) are mainly detected during S phase when encountered by the replication machinery [26, 27]. ICLs detected by the replisome initiate a complex set of enzymatic reactions that, as with many HR events in S phase, are regulated by the Fanconi Anaemia (FA) pathway [28]. Similarly, unrepaired base lesion encountered during DNA replication can be bypassed by the replisome and

DNA repair in mitosis

Early experiments showed that introducing DNA damage into early mitotic cells resulted in a transient reversal of mitosis [37]. However, until quite recently it was considered that once the decision to enter mitosis has been made DNA damage responses became largely irrelevant. It is now recognised that, when cell transit into mitosis, DNA damage responses are significantly modified. For example, in response to DSBs ATM activation and H2AX phosphorylation still occur (in fact more so because

Conclusion

The study of the mechanisms that regulate DNA repair and other DNA damage responses through the cell cycle is evolving rapidly. There is an immense amount of detail in the literature concerning the regulation of repair and checkpoint signalling [53••] by cell cycle dependent phosphorylation and ubiquitylation. In the next few years we will likely have a clearer understanding of how DNA repair pathway choice for DSBs, the most toxic of DNA lesions, is integrated with, and fine-tuned by, the cell

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgments

The authors acknowledge funding from the Wellcome Trust (110047/Z/15Z; AMC) and the Medical Research Council (MR/P018955/1; JMM). The funders have had no role in the preparation of this work or the decision to submit for publication.

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