Modification of chromatin in response to DNA damage

Once DNA damage has been detected, a series of events take place to signal to the damage repair pathways and initiate DNA repair. These complex multi-subunit machines carry out a number of tasks on nucleosomes including chemical modifications, histone exchange and sliding them on DNA (remodelling) (Figure 1) in what increasingly appears to be a coordinated process.

Recent work has begun to identify the components that make up these machines but their structures and mechanisms remain largely unknown. Structural studies of chromatin modifying complexes are limited to a few systems involved in remodelling. Although electron microscopy studies have revealed tantalising glimpses of the extraordinary shapes and architecture of the complexes, the mechanism of remodelling remains unclear. There are crystal structures of very few of the protein subunits of any chromatin modifying system and always of the protein in isolation rather than in the context of the complex.

Repair of DNA damage requires remodelling of nucleosomes to allow access by the DNA repair machinery and one chromatin remodelling complex (INO80) appears to be particularly involved in this process. The DNA damage response also results in covalent modification of nucleosomes which serves to recruit repair factors. For example, upon detection of DNA double-strand breaks, there is a rapid appearance of histone gamma-H2AX across a ~2Mb region around the break. The presence of gamma-H2AX serves to recruit a key regulator of the DNA damage response (MDC1) as well as several chromatin modifying complexes such as INO80, ySwr1, yNuA4 and hTIP60. One consequence of this recruitment is acetylation by complexes such as yNuA4 and hTIP60.

Acetylation of histones by yNuA4 stimulates further changes in chromatin, including the swapping of histone variants within nucleosomes catalysed by the ySwr1 complex. The ySwr1 complex replaces H2A with the variant Htz1 [16] although the consequences of this exchange remain unclear. In addition to a translocase subunit which is very closely related to INO80 protein, the ySwr1 complex also contains a number of other subunits in common with the INO80 complex such as actin, Arp4 (also found in yNuA4) and two RuvB-like proteins (Rvb1 & Rvb2) suggesting an overlap in some aspects of mechanism. However, the ySwr1 complex also contains a further half a dozen or so proteins that appear to be specific (Figure 2).

Figure 2. Overlapping core subunit composition of the INO80, ySwr1/hSRCAP, yNuA4 and hTip60 complexes. The overlaps are based on current understanding from a combination of sequence homologies and biochemical data were available and may be incomplete

In humans, the equivalent complex to ySwr1 is hSRCAP but, to complicate things further, several of the components of hSRCAP are also found in a larger complex called hTIP60 which also contains an acetylase equivalent to that in the yNuA4 complex. TIP60 also acetylates gamma-H2AX and exchanges it for H2AX. Hence, in humans, histone exchange and acetylase functions are combined within a single supra-molecular complex (hTIP60) consistent with the linked activities of ySwr1 and yNuA4. TIP60 has been shown to be localised at sites of DNA damage. 

Consistent with this role, hTIP60 complex associates with the INO80 complex, which in turn is associated with the Mre11/Rad50/Nbs1 complex that is required for double-strand break repair, bringing together the activities required for DNA damage processing and repair. 

The interconnectivity and interdependence of these components of the DNA damage response emphasises the benefits in understanding that will come from a holistic approach that encompasses all of these systems. Consequently, we are studying the structure and mechanism of complexes that perform three essential and coordinated functions of regulation of chromatin (remodelling, covalent modification and histone exchange) that are involved in the DNA damage response, which will lead to a major advance in our understanding of this key process.