Pathways and Mechanisms Governing Genome Duplication and Repair
Genome duplication is a crucial task for all cells, and its defects lead to tumorigenesis, aging, and developmental diseases. How a cell can faithfully duplicate its entire genome impinges heavily on processes that overcome naturally and frequently occurring replication blocks on DNA templates. These blocks manifest as DNA lesions, secondary structures, tightly bound protein complexes, or transcription-associated DNA-RNA hybrids. Emerging research suggests that replication is tightly coordinated with processes, such as DNA repair, protein modifications, and chromatin structure regulation, which assist replisomes in resuming DNA synthesis after obstacle removal or provide alternative synthesis strategies. We are just beginning to comprehend the intricate and dynamic interplay between these pathways.
Our lab has identified several important factors required for replication to be coordinated with other processes. The Smc5/6 complex is one of the three essential and highly conserved SMC (structural maintenance of chromosomes) complexes. More recently discovered than the other two complexes, cohesin and condensin, it is emerging as a multifunctional regulator of chromosomal integrity. As mutations in Smc5/6 subunits have been found in about 10 percent of cancer genomes sequenced thus far, understanding how the complex functions harbors important implications for diagnosing and treating cancers.
The architecture of the Smc5/6 complex and the crystal structure of Mms21 with an Smc5 domain
After our initial work of identifying the Smc5/6 complex in budding yeast, we have continued to delve deeper into its functions, interactions, and composition. We found that the 8-subunit Smc5/6 complex shares common structural features with the other SMC complexes, but unlike them, contains a subunit with SUMO ligase activity (Figure 1, Zhao 2005, Duan 2009a, 2009b). These studies provide the basis for understanding the assembly and architecture of this complex, as well as for developing accurate working models for the diverse roles this complex appears to play.
One of the functions of Smc5/6 is to regulate three branches of recombinational repair processes that assist replication on damaged DNA templates (Figure 2, Choi 2010). Within these pathways, Smc5/6 prevents the accumulation of joint DNA molecules, which can lead to chromosome breakage, fusions, and translocations.
A model for Smc5/6-mediated regulation of three recombinational repair pathways that allow replication completion when template DNA is damaged. Details in Choi 2010.
We have identified a physical interaction between Smc5/6 and the Mph1 DNA helicase (human FANCM homolog, mutated in Fanconi anemia patients), which is likely important for Smc5/6 to achieve this function (Chen 2009). Current work investigates how Smc5/6 influences Mph1 functions in conjunction with the Mph1-associated histone-fold proteins Mhf1 and 2. We are also examining how Smc5/6 affects the two Mph1-independent repair branches, such as how Smc5/6 uses its sumoylation activity to facilitate the resolution of joint DNA molecules and affects PCNA ubiquitination-mediated repair.
Our studies encompass both euchromatin and repetitive regions such as telomeres, rDNA, and centromeres (Figure 3; Zhao 2005, Takahashi 2008, Hang 2011, Yong-Gonzales 2013). These studies will provide a mechanistic understanding of how Smc5/6 orchestrates processes necessary to complete replication at different genomic regions.
Sumoylation in Signal Transduction and Dynamic Regulation of DNA Metabolism
mms21 mutations lead to the disruption of telomeric (green) and nucleolar (blue) structures
Modifying proteins with chemical groups or small proteins is a principal means of regulating biological processes on rapid time scale. SUMO is about 100 amino acids long and can be covalently linked to lysine residues of proteins in a process called sumoylation. Sumoylation requires the sequential action of three enzymes called SUMO E1, E2, and E3 (ligase) enzymes. This is a highly dynamic process and SUMO can be removed by specific proteases called desumoylation enzymes. Both sumoylation and desumoylation are essential for cell growth and affect diverse pathways. The addition and removal of SUMO can exert a dynamic range of effects on a protein’s functions, consequently serving as a switch or modulator of cellular processes.
We recently discovered that when cells are treated with agents mimicking chemotherapy drugs that induce DNA stress and replication blockage, several dozens of DNA metabolism proteins become sumoylated. We termed this novel response “DNA damage-induced sumoylation” (DDIS) (Cremona 2012). The scope of DDIS is similar to that of the well-known phosphorylation-based DNA damage checkpoint response; yet the two pathways target complementary sets of proteins.
Extensive DDIS acts in parallel with the DNA damage checkpoint to promote DNA replication and repair.
Such division of labor is also consistent with the observation that the two pathways operate largely independently and make separate contributions to cell survival (Figure 4). Results from several assays estimate that DDIS and checkpoint are equally important for genomic integrity. These features highlight the significance of DDIS and the potential efficacy of a two-pronged approach to cancer therapy simultaneously targeting both DNA response branches.
We are currently investigating how cells sense DNA stress and initiate the SUMO-based response. DDIS appears to be composed of multiple regulons controlled by different factors (Figure 4). We are uncovering the mechanisms underlying SUMO-based control of each of these different response modules and are revealing new paradigms about how sumoylation elicits broad effects on DNA replication and repair.
How DDIS is reversed when DNA stress is removed is another interesting question. Our previous work on a desumoylation enzyme and a SUMO-targeted ubiquitin ligase (STUbL) provides the basis for this aspect of the research (Figure 5, Zhao 2004, Burgess 2007, Palancade 2007). Finally, examining the crosstalk between DDIS and checkpoint will contribute greatly to a comprehensive view of the DNA damage response.
Ulp1 exhibits biased localization at the nuclear envelope — Ulp1 (green) is localized to a sub-region of the nuclear envelope (blue), which is not in contact with the nucleolus (red).
The yeast model system
Budding yeast, our experimental system of choice, provides a number of advantages for studying conserved eukaryotic pathways. The simplicity of the system, large number of established reagents and techniques, vast repository of information, and rapid experimental turnaround time have tremendously galvanized the discovery of new paradigms and pushed scientific boundaries. Through its use as a pioneer system for rigorous, in-depth molecular investigations, seminal contributions will continue to be made to the understanding of cancer- and aging-related mechanisms in human cells.
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