The histone protein H2AX is phosphorylated rapidly over large regions of DNA around sites of DSBs by ATM kinase (Burma and others 2001). Loss of H2AX phosphorylation occurs rapidly with the repair of DSBs, but the biochemical details of dephosphorylation remain to be ascertained. A recent study showed that in human cells, a background level of H2AX phosphorylation occurred in about 5% of the cells. After low doses of X-rays that initially increased the level to 10%, most cells eliminated this phosphorylation, except for a small fraction in which it persisted unless the cell entered DNA synthesis (Rothkamm and Lobrich 2003). Whether this means that a small fraction of cells cannot repair some classes of LMDS or that dephosphorylation of H2AX can be slower than repair itself in a subset of cells remains to be determined.
The DNA-PK kinase is a member of a class of phosphatidyl-3-inosityl enzymes that includes ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-related (ATR) kinases, all of which are involved in signaling the presence of DNA damage (Shiloh 2001, 2004; Figure 1-11). Although DNA-PK kinase can phosphorylate many proteins in vitro, it is unclear which proteins it usually phosphorylates in vivo. Early cytologic evidence of X-ray damage is phosphorylation of a histone protein to create γ-H2AX foci that are visible microscopically within minutes of irradiation.
After the rapid phase of rejoining is complete, the repair of DSBs slows to a second phase with a half-time of several hours. Foci containing the hMre11/hRad50/Nbs complex form or persist and reach a maximum at about 4–6 h. Because this complex has endonuclease and DNA-binding activity, it may be involved in the slower repair of refractory DSBs that cannot be repaired by the earlier, fast mechanism. The complex is not active unless the Nbs1 protein is phosphorylated on several sites by ATM kinase (Figure 1-11), which is itself activated by DNA breaks (Shiloh 2001; Bakkenist and Kastan 2003). The precise DNA structures involved in these refractory breaks are unknown. However, one model suggests that nuclease action by the Mre11 complex resects single DNA strands and that short regions of sequence identity (microhomologies) can be used for alignment and rejoining of DNA strands (Figure 1-12).
Repair of a DSB by HR involves matching the two broken ends of a DNA strand with identical sequences of intact DNA (Figure 1-12). The broken and intact molecules are aligned according to their sequences and encompassed by a toroid of hRad51 molecules that facilitate repair by having DNA single strands invade their homologues, producing an X-shaped four-armed structure called a Holliday junction. Resolution of this structure by specific junction nucleases produces two intact double-strand DNA molecules with or without exchanges according to the orientation of the resolution nuclease actions. The activity of hRad51 is enhanced by other factors, such as hRad52, XRCC2, and XRCC3, and suppressed by p53, which binds to both Holliday junctions and hRad51 (Buchhop and others 1997). HR is much more efficient and important for repair in yeast and somatic chick cells than in normal (nonmalignant) mammalian (human) somatic cells, where NHEJ is the dominant mechanism for DSB repair (Sonoda and others 1998). However, there are exceptions, and there may be times in the cell cycle, such as late S, when HR assumes greater importance because of the proximity of sister chromatids (Thompson 1996). The low level of sister-chromatid exchange, a form of HR, induced by X-rays and high-LET radiation indicates that, in absolute terms, HR remains a minor pathway for the repair of damage caused by ionizing radiation in somatic cells.
There is some question about the source of an identical matching sequence for repair by HR in somatic human cells. A homologous sequence may be the other allele on a chromosome of a recently replicated sister-chromatid sequence on a daughter chromatid or a similar sequence in a repetitive region along the same chromosome. In the latter case the sequences may not be identical over long regions, and the mechanism is known as “homeologous” recombination. Recombination between alleles on separate chromosomes occurs at much lower frequency than between identical sequences on sister chromatids or arranged in tandem on the same chromosome. In general, HR between sister chromatids may occur at higher frequencies late in the cell cycle (e.g., late S; Thompson and Schild 1999), and homeologous recombination is likely to result in the loss of intervening sequences with the production of deletion mutations.
The HR involving hRad51 can be visualized immuno-histochemically: foci containing hRad51, Brca1, and other proteins can be seen microscopically soon after irradiation (Scully and others 1997). Cells generally exhibit either hRad51 foci or hMre11/hRad50/Nbs foci, but not both, and the choice of which of the mutually exclusive pathways an irradiated cell follows may be determined by Brca1 (Parvin 2001).
Bacteria live in a highly variable environment and have evolved efficient inducible DNA repair processes to deal with sudden challenges of DNA damage from oxygen free radicals, ionizing radiation, chemicals, and ultraviolet radiation. These inducible repair pathways are now mechanistically well understood. In Escherichia coli, the regulatory genes soxR, ada, and lex control transcription of DNA repair functions, and increased amounts of relevant DNA repair enzymes can be produced in response to environmental challenges. In mammalian cells, the same types of DNA damage are recognized by similar DNA repair enzymes. However, a