Damage site chromatin: open or closed?

Abstract

Technical advances in recent years, such as laser microirradiation and chromatin immunoprecipitation, have led to further understanding of DNA damage responses and repair processes as they happen in vivo and have allowed us to better evaluate the activities of new factors at damage sites. Facilitated by these tools, recent studies identified the unexpected roles of heterochromatin factors in DNA damage recognition and repair, which also involves poly(ADP-ribose) polymerases (PARPs). The results suggest that chromatin at damage sites may be quite structurally dynamic during the repair process, with transient intervals of 'closed' configurations before a more 'open' arrangement that allows the repair machinery to access damaged DNA.

Figure 1. In vivo tools to study DNA damage recognition and response pathways

A. ChIP analysis of endonuclease-induced DSBs. The HO endonuclease in yeast, and the I-SceI and I-PpoI homing endonucleases in mammalian cells, have been used to induce DSBs. While HO and I-PpoI cut endogenous recognition sequences, the I-SceI recognition sequence is introduced into the genome by stable transfection. Factor binding or modification in the vicinity of DSB sites can be analyzed by ChIP using specific PCR primers. B. Fluorescent microscopy analysis following nuclear microirradiation. Selective sub-nuclear exposure of UVC using micropore filters has been used to study the NER pathway. Focused laser microbeams (of various wavelengths, energies, etc.) or charged particles, delivered to sub-micron regions in the cell nucleus (with or without DNA sensitization), have been used mainly to study DSBs. However, other types of damage are also induced, including CPD, SSBs, and in some cases, base damage. In addition, photo-activation of psoralen derivatives by microirradiation induces interstrand crosslinking damage (ICLs).

Figure 2. Recruitment of heterochromatic factors to DNA damage sites

PARP is activated and rapidly recruited to the DNA damage sites resulting in the accumulation of PAR in the vicinity of the damage sites. PAR serves as a landing pad for macroH2A, polycomb repressor complex(es), and NuRD. The PRC recruitment is accompanied by histone H3K27me3. In addition, both HDAC1 and HDAC2, as well as HP1, are also recruited to the damage sites, although the recruitment mechanism(s) is unclear. These heterochromatin/gene silencing factors may be important for local transcriptional silencing at the damage sites, dictating DNA repair pathway choice, and/or recruiting DNA repair factors, such as 53BP1 and BRCA1, to promote efficient DNA repair.