Cancer and many genetic deseases are associated with genetic instability and cell cycle deregulation. The maintenance of genome integrity and proper cell cycle progression relies on a plethora of mechanisms that coordinate DNA repair and chromosome dynamics during the cell cycle. Our research is focused in understanding the molecular base of these mechanisms, paying particular attention to the role that chromatin dynamic plays in the generation and repair/tolerance of DNA lesions. In particular, our group is focused in the following research lines:
1. Role of chromatin assembly on genome integrity and cell cycle progression.
Maintaining the stability of the replication forks is one of the main tasks of the DNA damage response. Specifically, checkpoint mechanisms detect stressed forks and prevent their collapse. Using the yeast Saccharomyces cerevisiae we have shown that defective chromatin assembly in cells lacking either H3K56 acetylation or the chromatin assembly factors CAF1 and Rtt106 affects the integrity of advancing replication forks, despite the presence of functional checkpoints. This loss of replication intermediates is exacerbated in the absence of the recombination protein Rad52, suggesting that collapsed forks are rescued by homologous recombination and providing an explanation for the accumulation of recombinogenic DNA damage displayed by these mutants (Clemente-Ruiz et al. 2011).
These phenotypes mimic those obtained by a partial reduction in the pool of available histones and are consistent with a model in which defective histone deposition uncouples DNA synthesis and nucleosome assembly, thus making the fork more susceptible to collapse (Clemente-Ruiz and Prado 2009). Our findings reveal that correct nucleosome positioning is required for replication fork stability (revised in Prado and Clemente-Ruiz 2012) (Figure 1). We are currently analyzing the mechanisms by which nucleosome assembly prevents fork collapse and genetic instability, as well as the impact that defective histone deposition has on genome integrity in human cell lines.
2. Histone H2A-by-H2A.Z (Htz1) replacement and genome integrity. Cells have evolved many different mechanisms by which chromatin is modified along the cell cycle to regulate DNA metabolism. Cells can replace canonical histones with variants that specifically alter chromatin structure. One such variant, H2A.Z – Htz1 in yeast –, is an evolutionary conserved histone with roles in transcription, silencing, genome integrity and cell cycle progression. Htz1 is incorporated into chromatin by SWR1, an ATP-dependent remodeling complex. We are interested in understanding how SWR1 and Htz1 prevent genetic instability. We have obtained new insights in the mechanism by which SWR1 replaces H2A with Htz1, and shown that the remodeling activity of SWR1 can be highly genotoxic in the absence of Htz1 (Morillo-Huesca et al. 2010) (Figure 2), further supporting the notion that a strict control of chromatin dynamic is required to maintain genome integrity.
3. Mechanisms and cell cycle regulation of the DNA damage tolerance. The DNA damage response (DDR) functions as a barrier that prevents genetic instability associated with cancer development. The integrity of the replication fork is a central component of this response, and mutations that affect the mechanisms involved in the detection, stabilization, and repair of stressed forks cause genome instability. In every cell cycle, endogenous and exogenous agents cause DNA lesions that impair the advance of the replication forks (e.g., the exposure to UV light or alkylating agents). This uncouples DNA unwinding and synthesis, thus leading to an accumulation of single-stranded DNA (ssDNA). To complete replication, cells are able to restart DNA synthesis downstream the DNA damage leaving a ssDNA gap behind the fork. Therefore, an important part of the DDR is aimed at promoting DNA replication past DNA lesions and repairing the ssDNA gaps generated during this process. This response of DNA damage tolerance (DDT) is thereby essential for cell cycle progression and genome integrity. The mechanisms of DDT are conserved from yeast to humans, even though they are better understood in yeast.
The ssDNA gaps are repaired by translesion synthesis (TLS) and error-free post-replicative repair (PRR) mechanisms. Whereas the former fills the gap extending the 3’-end past the damaged template using specialized DNA polymerases able to incorporate a nucleotide opposite the lesion, the later uses the information of the sister chromatid through a template switch (TS) mechanism. Consequently, the choice between the error-prone TLS and the error-free TS determines whether the repair is or not mutagenic and, therefore, it is a key determinant in cancer development. We are particularly interested in understanding the role that homologous recombination proteins play in DDT and how their activities are coordinated with other DDT components during the cell cycle. We have shown that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non-repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non-DSBs DNA lesions, presumably single-stranded DNA gaps, which are generated during the replication of alkylated DNA. The repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for homologous recombination where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle–regulated replicative and repair functions (Gonzalez-Prieto et al. 2013) (Figure 3).