Maintenance of genome stability

By Kate Dry & Steve Jackson *

Read about the current knowledge and recent findings in the DNA damage response pathway.

Cellular responses to DNA damage pathway

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The DNA in our cells is under constant attack by exogenously- and endogenously-arising DNA-damaging agents such as ultraviolet light, mutagenic chemicals and ionizing radiation. It is estimated that a single cell can encounter approximately 100,000 DNA damaging events per day. Consequently cells have evolved elaborate damage response mechanisms to repair damaged DNA to prevent the lesions being passed on to daughter cells. The most deleterious type of damage is the double-strand break (DSB) where both strands of the DNA double helix are simultaneously broken and the DNA ends become physically dissociated from one another. Proper repair of chromosomal DSBs is critical for maintaining cellular viability and genomic integrity and, in multicellular organisms, for suppression of mutagenesis, and the prevention of mutations leading to the generation of unstable chromosomes, which could lead to cancer and other diseases.

Over recent years there has been much progress in our understanding of how cells detect, signal and repair DNA DSBs (Jackson, 2002). However, new players in the DNA damage response (DDR) are still being identified that add to the complexity of this system and whose functions we are only just beginning to understand. For example, in mammalian cells DSBs are detected by several factors including the Mre11-Rad50-Nbs1 (MRN) complex, Ku and three related proteins: DNA-PKcs, ATM and ATR. These latter three proteins are all members of the PIKK family of protein kinases and are able to signal the presence of DNA damage to the cell via phosphorylation of downstream target proteins. In addition to the proteins that sense damaged DNA, this and other labs have identified novel proteins that act as adaptors or facilitators of the DNA damage response and are required for efficient activation and signaling of the DNA damage. For example, we identified the human protein, MDC1, as a binding partner for the MRN complex, recruiting the MRN complex to chromatin at sites of DNA damage and acting as a mediator in the initial detection and signaling of DNA damage (Goldberg et al, 2003). Furthermore we have shown that the PIKK partner proteins Nbs1, ATRIP and Ku80 contain a common evolutionarily conserved motif that is required for their effective interaction with their PIKK, ATM, ATR or DNA-PKcs respectively (Falck et al, 2005). Additionally, these motifs are necessary for the recruitment of these PIKKs to sites of DNA damage and for downstream PIKK-dependent signaling events that lead to cell cycle checkpoint arrest and the efficient repair of damaged DNA. How these and other recently identified proteins such as Rif1, PTIP, Claspin and RNF8 interact, function and are regulated in order to modulate the DNA damage response is the focus of much current research (for recent reviews see Nyberg et al., 2002; Khanna and Jackson, 2001).

Additional factors such as 53BP1 and BRCA1 are also recruited to sites of damage and modulate and amplify the PIKK-dependent signals, leading to recruitment of repair factors and phosphorylation of downstream targets such as the checkpoint kinase Chk2. The checkpoint kinases Chk1 (phosphorylated by ATR) and Chk2 (phosphorylated by ATM) then target effector proteins involved in modulating DNA repair, transcription and cell-cycle control (Kastan and Bartek). This signaling pathway thus arrests actively dividing cells to allow sufficient time to repair the DNA damage. Alternatively, if the damage is excessive or irreparable, signals activate the apoptotic pathway via p53, ultimately leading to cell death, or trigger long-term growth arrest called senescence. Following DNA damage signaling and cell cycle arrest, repair factors are recruited to sites of damage. There are two major pathways in mammalian cells for the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR) (Lieber et al., 2003; West, 2003; Sung and Klein, 2006). These pathways act in a mainly separate but complementary manner to bring about DNA repair. HR functions primarily to repair lesions at the replication fork and in G2, using the sister chromatid as a template to carry out error-free repair. NHEJ repairs DSBs at all stages of the cell cycle, bringing about the ligation of two DNA DSBs without the need for sequence homology and so is error-prone.

The NHEJ pathway in mammals requires DNA-PK, a multi-subunit protein comprising three components: Ku70 and Ku80 which together form a DNA end-binding complex (Ku), and the catalytic subunit DNA-PKcs (Gottlieb et al., 1993; Smith and Jackson, 1999). The Ku proteins bind DNA ends of DSBs and recruit other NHEJ factors such as DNA-PKcs to these ends, leading to activation of DNA-PKcs. Work in our lab has contributed to our knowledge of how DSBs are repaired by NHEJ both in mammalian cells (e.g. Critchlow et al, 1997) and through the identification of functional homologues in yeast (e.g. Boulton and Jackson, 1996; Teo et al., 2000). Furthermore, we recently identified XLF (XRCC4-like factor) as another member of the NHEJ repair complex that is involved in the ligation of DNA ends along with DNA ligase IV and XRCC4 following the processing of broken ends by other proteins such as the MRN complex and Artemis (Ahnesorg et al., 2006).

Alternatively, DSBs can be repaired by HR. HR is the preferred mechanism of DSB repair in yeast and consequently much of what is known about this pathway has come from studies in this model organism. HR is a complex process that initially requires the resection of the DNA DSB by processes involving the MRN complex to expose 3’ single stranded DNA tails (Lavin, 2004; D’Amours and Jackson, 2002). These are then bound by a number of proteins including Rad50, Rad51, Rad52, Rad54 and RPA. The nucleoprotein filament then interacts with an undamaged DNA molecule leading to strand-exchange events, DNA synthesis by DNA polymerases and DNA ligation. In addition, the breast cancer susceptibility proteins BRCA1 and BRCA2 have been found to play a role in HR (Venkitaraman, 2001).

Finally it is important to remember that DNA in eukaryotes is densely packaged into chromatin, and how cells signal and repair damaged DNA in this context is only now being explored. For example, the variant histone H2AX is phosphorylated in response to DNA damage and then binds to MDC1 (Stucki et al., 2005), while 53BP1 is known to bind to methylated histone H3 at sites of DNA damage (Huyen et al., 2004). Recent work has linked the DDR to other chromatin modifications, such as ubiquitination and sumoylation, and chromatin modifying complexes involving proteins such as Tip60 (Downs et al., 2007). In addition, we do not yet know a great deal about how the DNA damage response is switched off once repair has been completed and what signals are required to restore cell cycle progression. DDR proteins also function in other cellular events including the control of telomere structure and at abnormal telomeres (d’Adda di Fagagna et al., 2004), in the generation of antibodies via V(D)J recombination (Bassing and Alt, 2004) and during meiotic recombination (Keeney and Neale, 2006). These and other areas will be the focus of much research for many years to come.

References

1. Ahnesorg P, Smith P, Jackson SP. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell. 2006 Jan 27;124(2):301-13.
2. Bassing CH, Alt FW. The cellular response to general and programmed DNA double strand breaks. DNA Repair (Amst). 2004 Aug-Sep;3(8-9):781-96. Review.
3. Boulton SJ, Jackson SP. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res. 1996 Dec 1;24(23):4639-48.
4. Critchlow SE, Bowater RP, Jackson SP. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr Biol. 1997 Aug 1;7(8):588-98.
5. d'Adda di Fagagna F, Teo SH, Jackson SP. Functional links between telomeres and proteins of the DNA-damage response. Genes Dev. 2004 Aug 1;18(15):1781-99. Review.
6. D'Amours D, Jackson SP. The Mre11 complex: at the crossroads of dna repair and checkpoint signaling. Nat Rev Mol Cell Biol. 2002 May;3(5):317-27. Review.
7. Downs JA, Nussenzweig MC, Nussenzweig A. Chromatin dynamics and the preservation of genetic information. Nature. 2007 Jun 21;447(7147):951-8.
8. Falck J, Coates J, Jackson SP. Conserved modes of recruitment of ATM, ATR and DNAPKcs to sites of DNA damage. Nature. 2005 Mar 31;434(7033):605-11.
9. Goldberg M, Stucki M, Falck J, D'Amours D, Rahman D, Pappin D, Bartek J, Jackson SP. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature. 2003 Feb 27;421(6926):952-6.
10. Gottlieb TM, Jackson SP. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131-42.
11. Huyen Y, Zgheib O, Ditullio RA Jr, Gorgoulis VG, Zacharatos P, Petty TJ, Sheston EA, Mellert HS, Stavridi ES, Halazonetis TD. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature. 2004 Nov 18;432(7015):406-11.
12. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002 May;23(5):687-96.
13. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature. 2004 Nov 18;432(7015):316-23.
14. Keeney S, Neale MJ. Initiation of meiotic recombination by formation of DNA doublestrand breaks: mechanism and regulation. Biochem Soc Trans. 2006 Aug;34(Pt 4):523-5. Review.
15. Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet. 2001 Mar;27(3):247-54. Review.
16. Lavin MF. The Mre11 complex and ATM: a two-way functional interaction in recognising and signaling DNA double strand breaks. DNA Repair (Amst). 2004 Nov 2;3(11):1515-20. Review.
17. Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism and regulation of human nonhomologous DNA end-joining. Nat Rev Mol Cell Biol. 2003 Sep;4(9):712-20.
18. Nyberg KA, Michelson RJ, Putnam CW, Weinert TA. Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet. 2002;36:617-56. Review.
19. Smith GC, Jackson SP. The DNA-dependent protein kinase. Genes Dev. 1999 Apr 15;13(8):916-34. Review.
20. Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 2005 Dec 29;123(7):1213-26.
21. Sung P, Klein H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol. 2006 Oct;7(10):739-50. Epub 2006 Aug 23. Review.
22. Teo SH, Jackson SP. Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks. Curr Biol. 2000 Feb 10;10(3):165-8.
23. Venkitaraman AR. Functions of BRCA1 and BRCA2 in the biological response to DNA damage. J Cell Sci. 2001 Oct;114(Pt 20):3591-8. Review.
West SC. Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol. 2003 Jun;4(6):435-45. Review.


Kate Dry and Steve Jackson
Wellcome Trust/Cancer Research UK Gurdon Institute
Cambridge, UK

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