4.2 Chromatin architecture in mammalian cells
4.2.2 Chromatin structure during DNA replication and DNA damage repair
of chromatin rearrangement has significant impact on the accessibility of DNA, especially for proteins that directly bind to DNA. Binding of transcription factors and DNA repair proteins to their DNA substrates, as well as replication fork progression are two examples of crucial processes that are significantly affected by chromatin structure. On the scale of the 10 nm fiber, nucleosomes have to be slid along the DNA or even completely removed from the fiber to allow other proteins access to the DNA. Furthermore, chromatin architecture needs to be restored after the completion of DNA-dependent processes to
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avoid unfavorable or unscheduled actions on DNA. Nucleosome dynamics can be affected in at least three different ways: (1) DNA methylation which affects DNA topology and stability and thereby also DNA interaction with histones and other proteins (Bartke et al, 2010; Collings et al, 2013), (2) post-translational modifications of N-terminal histone tails and of histone core domains which affect the histone’s affinity towards DNA and DNA-remodeling factors (Mersfelder & Parthun, 2006), and (3) ATP-dependent chromatin remodeling complexes which slide complete nucleosomes or disassemble histone octamers from the chromatin fiber (Bowman, 2010). In addition, there is crosstalk between these different remodeling pathways leading to a complex network of chromatin remodeling activities. In the following, two important DNA-dependent processes – DNA replication and DNA damage repair – will be outlined with respect to chromatin remodeling necessary for their effective operation.
4.2.2.1 Chromatin structure and DNA damage repair
One vital DNA-dependent process that relies on chromatin reorganization is the repair of DNA lesions. The scale of chromatin remodeling required for effective damage repair depends on the nature and the extent of the damage. The eukaryotic cell has evolved highly specialized DNA repair pathways for all kinds of DNA lesions and each of these pathways comprises varying chromatin remodeling processes. How chromatin structure is affected by DNA repair and vice versa will be described here exemplarily for the repair of DNA strand breaks (DSBs). Many of the processes described in this context also apply of other repair pathways (reviewed in (Cann & Dellaire, 2011; Lafon-Hughes et al, 2008; Sulli et al, 2012)).
When DNA is lesioned in a way that breaks covalent bonds within the DNA backbone, the cell needs to react instantaneously to ensure genome integrity. To this end, chromatin has to relax to allow repair factors to gain access to the damage (Cann & Dellaire, 2011; Ziv et al, 2006). How exactly DSBs are recognized and which structure or topology change leads to the initiation of the following cascade of repair events is still not completely understood.
So far, the earliest detectable reaction after induction of DSBs is the PARylation of N-terminal histone tails of core histones and of multiple other acceptor proteins at or near the damage site. The consequences of PAR formation at the site of DNA damage are multiple and diverse. They range from chromatin remodeling (interestingly, both, chromatin condensation and decondensation can be triggered by PARylation), to protein recruitment and protein retention. This mixture of even counteractive effects points to a highly dynamic process which is dependent on timing and location of PAR formation and degradation. One of the first initial steps of DSB repair is also the activation of the key kinase ATM (ataxia telangiectasia mutated). Activated ATM phosphorylates the histone variant H2AX at S139 (then designated γH2AX) directly at the damage site but also in
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vicinity surrounding region of up to several megabases up- and downstream of the damage (Rogakou et al, 1999). Additionally, H2AX can be phosphorylated by other kinases which also can be activated in the course of DSB repair: the ATM-related kinases ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase). γH2AX then serves as a binding platform for many sensor proteins which – once recruited – are often targets for phosphorylation themselves and are required for subsequent DNA repair steps. One of these sensors was shown to recruit ATP-dependent chromatin remodelers to the site of DNA damage (van Attikum et al, 2007). Other histone modifications that directly affect chromatin structure and composition in response to DSB induction are methylation and acetylation of histone tails. Especially acetylation is associated with local chromatin relaxation. Additionally, histone tail acetylation enhances ATM activity establishing a positive feedback loop, (Bhoumik et al, 2008; Sun et al, 2005). DSBs also lead to local recruitment of histone deacetylases (HDACs), a finding that further supports the concept of a highly dynamic rearrangement of the chromatin fiber in response to DSBs. Underscoring these dynamics, the recruitment of HDACs was recently demonstrated to favor DSB repair via non-homologous end joining with respect to homologous recombination. This observation clearly shows that DNA damage not only causes massive changes in chromatin architecture, but chromatin structure in turn affects the manner a DSB is repaired (Lukas et al, 2011b; Miller et al, 2010).
4.2.2.2 Chromatin structure and DNA replication
A eukaryotic cell cycle comprises different phases. During G1 phase, a cell prepares for genome replication which takes place during S phase. In the following G2 phase, the entire genome is completely duplicated and distributed equally between two daughter cells during mitosis (or M phase) which then reside again in G1 phase. In the course of this cycle, all chromosomes need to be decondensed in a timely fashion so that the replication machinery can produce a copy of the entire genome. After completed replication the chromosomes recondense again. To enable replisome progression, the chromatin fiber has to be disassembled and the DNA helix unwound in front of the replication fork. Given an approximate replication speed of 2 to 3 kb per minute (Méchali, 2010), 10 to 15 nucleosomes have to be removed from the chromatin fiber every minute to guarantee effective replication fork progression (Alabert & Groth, 2012). First, the linker histone H1 which stabilizes the 10 nm fiber has to be removed before nucleosome dismantling.
Dissociation of H1 from the nucleosome is induced by its phosphorylation by the S phase-CDK1 (cyclin-dependent kinase 1) which is activated by CYCA (cyclin A) (Thomson et al, 2010). The following disassembly of nucleosomes ahead to the replication fork takes place only in very close proximity to the fork and is therefore thought to be a direct consequence of a collision of the replicative helicase with a nucleosome. Whether other proteins are involved in nucleosome disruption is so far unknown. Histones that are evicted in front of
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the replication fork are not simply lost: they are precious raw material for the condensation of the newly synthesized DNA daughter strands. By passing on old histones – including their post-translation modification pattern which was termed the “histone code” – the cell does not only economize but passes on valuable information in addition to the DNA sequence. This information is prerequisite for re-establishing the proper chromatin structure in the daughter cells. The current model of redistribution of parental histones to daughter DNA strands suggests that the parental H3/H4-tetramers are randomly integrated into the new strands where they are combined with either new or old H2A/H2B-dimers (Ransom et al, 2010; Xu et al, 2010). The shuttling of old histones from ahead of the fork to the newly synthesized DNA strands is aided by specialized histone binding proteins. So-called histone chaperones bind to histones or histone multimers and form complexes with components of the replisome, e.g. the replicative helicase (Jasencakova et al, 2010). The so far best studied histone chaperones which directly impact on replication efficiency are ASF1 (anti-silencing function protein 1), FACT (facilitates chromatin transcription) (Bao
& Shen, 2006; Formosa, 2012) and CAF1 (CCR4-associated factor 1). The different histone chaperones either display preferences for binding a specific histone or have multiple histone binding partners. The proportion of new and old histones bound to these chaperones strongly depends on the processivity of the replisome. When replication is disturbed more old histones are “stored” in complex with histone chaperones. A molecular mechanism that explains how histone chaperones escort their histone or histone multimer from ahead of the fork to the other side of the replisome remains to be elucidated.