Smc5/6 and replication fork stability
Lead Research Organisation:
University of Sussex
Department Name: Brighton and Sussex Medical School
Abstract
The accurate inheritance of the genetic material (DNA) is key to the survival of all organisms. DNA has to be copied (replicated) before cells divide but obstacles such as damaged DNA bases and DNA-proteins complexes can lead to replication stalling and breaking down. Cells have developed a range of mechanisms to remove such obstacles and if the DNA damage cannot be removed it can be bypassed either during or immediately after DNA copying by a range of mechanisms. This is important for survival but can be at the expense of an increased error rate leading to mutations and an increase in genome rearrangements. Since such changes can lead to cancer it is vital that copying is coordinated with repair and restarts correctly after stalling.
The Smc6 protein is essential for cell survival in all organisms from yeast to humans. Our work in yeast suggests that Smc6 has an important role in coordinating the repair of DNA damage with the copying of the DNA which must be accurate so that each new cell contains identical genetic material. When Smc6 is not fully functional cells use inappropriate processes to repair or tolerate the DNA damage and this leads to an increase in copying errors, which can result in changes to the genome and sometimes to cell death.
We want to understand how Smc6 acts to protect us from the deleterious effects of DNA damage during DNA copying because this has implications for the development of cancer and aging in the general population. Because yeast cells repair and copy their DNA in a similar way to human cells, and because yeast is easy and cheap to manipulate, we can use yeast to study how the Smc6 protein associates with the DNA copying apparatus and the DNA itself, and establish if and how it coordinates the activity of other factors to ensure copying restarts correctly.
The Smc6 protein is essential for cell survival in all organisms from yeast to humans. Our work in yeast suggests that Smc6 has an important role in coordinating the repair of DNA damage with the copying of the DNA which must be accurate so that each new cell contains identical genetic material. When Smc6 is not fully functional cells use inappropriate processes to repair or tolerate the DNA damage and this leads to an increase in copying errors, which can result in changes to the genome and sometimes to cell death.
We want to understand how Smc6 acts to protect us from the deleterious effects of DNA damage during DNA copying because this has implications for the development of cancer and aging in the general population. Because yeast cells repair and copy their DNA in a similar way to human cells, and because yeast is easy and cheap to manipulate, we can use yeast to study how the Smc6 protein associates with the DNA copying apparatus and the DNA itself, and establish if and how it coordinates the activity of other factors to ensure copying restarts correctly.
Technical Summary
Replication fork stability is vital for cell and organismal survival. Homologous recombination (HR) rescues collapsed forks, but at the potential expense of genome instability. In humans HR proteins are essential for S phase progression and defects in regulating HR, i.e. in Bloom?s syndrome, lead to increased cancer susceptibility. This underscores the importance of HR pathways for human health. We recently showed that the essential (and highly conserved) Smc5/6 complex is required to recruit HR proteins to stable stalled replication forks.
SMC complexes, including Cohesins and Condensins, are required for higher order chromosome structure and segregation. The Smc5/6 complex is the least well understood. Phenomenologically, Smc5/6 is required for HR, rDNA stability and telomere maintenance. By characterising Schizosaccharomyces pombe smc6 mutants, we defined two separate Smc5/6 functions in HR. First, most smc6 mutants are defective in processing recombination-dependent DNA intermediates when replication forks collapse. This leads to defects in chromosome segregation and increased rDNA recombination: a ?late? role in HR.
Second, we showed that Smc5/6 is required to load Rpa and Rad52 when replication stalls and so keep such stalled forks primed for resumption. This new ?early? role for Smc5/6 is defective specifically in the smc6-74 mutant, which maps to a conserved ?arginine finger?. In bacterial Smc proteins this domain is required for DNA-dependent ATP hydrolysis, likely to regulate opening and closing of Smc head domains. Defects in the early role correlated with increased rDNA stability. Thus Smc5/6 regulation of HR is important for replication resumption when forks are stalled but comes at the expense of increased genome instability. This function is particularly important in regions of unidirectional replication.
This proposal seeks to define the mechanism by which Smc5/6 regulates HR at stalled forks. We will determine:
? If DNA-dependent ATP hydrolysis is required for fork stability. Smc5/6 complexes will be purified and ATP hydrolysis analysed in relevant mutants.
? If other Smc5/6 domains/enzymatic activities are required. Smc6 mutants that suppress rDNA recombination will be identified, their responses to DNA damage and their ATPase activity analysed.
? Whether Smc5/6 functions directly or indirectly. Smc5/6 has been implicated in loading cohesins after DNA damage. The chromatin loading of the other Smc complexes upon replication stalling will be characterised in relevant smc6 mutants.
? If previously active replication forks resume replication efficiently (genome-wide and at the rDNA) or if new replication origins fire to complete S phase in smc6 mutants.
SMC complexes, including Cohesins and Condensins, are required for higher order chromosome structure and segregation. The Smc5/6 complex is the least well understood. Phenomenologically, Smc5/6 is required for HR, rDNA stability and telomere maintenance. By characterising Schizosaccharomyces pombe smc6 mutants, we defined two separate Smc5/6 functions in HR. First, most smc6 mutants are defective in processing recombination-dependent DNA intermediates when replication forks collapse. This leads to defects in chromosome segregation and increased rDNA recombination: a ?late? role in HR.
Second, we showed that Smc5/6 is required to load Rpa and Rad52 when replication stalls and so keep such stalled forks primed for resumption. This new ?early? role for Smc5/6 is defective specifically in the smc6-74 mutant, which maps to a conserved ?arginine finger?. In bacterial Smc proteins this domain is required for DNA-dependent ATP hydrolysis, likely to regulate opening and closing of Smc head domains. Defects in the early role correlated with increased rDNA stability. Thus Smc5/6 regulation of HR is important for replication resumption when forks are stalled but comes at the expense of increased genome instability. This function is particularly important in regions of unidirectional replication.
This proposal seeks to define the mechanism by which Smc5/6 regulates HR at stalled forks. We will determine:
? If DNA-dependent ATP hydrolysis is required for fork stability. Smc5/6 complexes will be purified and ATP hydrolysis analysed in relevant mutants.
? If other Smc5/6 domains/enzymatic activities are required. Smc6 mutants that suppress rDNA recombination will be identified, their responses to DNA damage and their ATPase activity analysed.
? Whether Smc5/6 functions directly or indirectly. Smc5/6 has been implicated in loading cohesins after DNA damage. The chromatin loading of the other Smc complexes upon replication stalling will be characterised in relevant smc6 mutants.
? If previously active replication forks resume replication efficiently (genome-wide and at the rDNA) or if new replication origins fire to complete S phase in smc6 mutants.