Investigating the role of DNA2 and homologous recombination in the recovery of stalled DNA replication forks
Lead Research Organisation:
University of Sussex
Department Name: Sch of Life Sciences
Abstract
Before every cell division, cells face the enormous task of producing a faithful copy of their genome so that both daughter cells can receive the correct complement of DNA. To do this, the two strands of the DNA duplex are unwound and copied at sites of DNA replication called DNA replication forks. These forks need to travel the full length of the genome, but their passage along chromosomes is often impeded as they meet obstacles and stresses. Cancer cells, with their fast and poorly regulated cell division cycle, experience fork stalling more frequently than normal cells. Counteracting fork stalling, cells employ an arsenal of factors to ensure that the forks restart to complete DNA replication. If this is not accomplished before cell division, the tearing apart of unreplicated segments of DNA can cause the kind of chromosomal aberrations seen in cancer cells. It is therefore of great fundamental biological interest and biomedical importance to understand the interplay of factors that mediate the recovery of stalled replication forks.
One prominent pathway of replication fork recovery is homologous recombination. This pathway is particularly important when the conventional replication machinery dissociates from the fork. Homologous recombination proteins can assemble a surrogate DNA synthesis machinery that promotes the continuation of replication. However, recombination-dependent DNA synthesis copies DNA less faithfully and can also lead to mutations and chromosomal changes. It is currently not clear how cells decide when to deploy homologous recombination at stalled replication forks and how they balance the risks and benefits associated with this pathway. It seems likely that cells only use this pathway when necessary and preferentially use mechanisms that directly restart the fork retaining the conventional replication machinery, hence avoiding the risk of mutations. One factor implicated in this direct form of fork restart is DNA2. Highlighting its biological significance, cells are unable to survive without DNA2 and its dysfunction results in a congenital dwarfism disorder known as Seckel syndrome. Elevated DNA2 levels on the other hand have been observed in cancer and it is thought DNA2 helps cancer cells to survive frequent replication fork stalling. We find that experimental degradation of DNA2 results in unusually high levels of homologous recombination DNA synthesis at stalled replication forks and cells enter a permanently non-dividing state. This not only explains why DNA2 is essential, but also provides a unique opportunity to determine how other fork restart pathways work alongside DNA2, and how the complex network of restart factors cooperates to choose the most appropriate mechanism to restart forks. Thus, our analysis will elucidate how extensively cells deploy homologous recombination at stalled replication forks and which regions of the genome are most vulnerable to unwarranted homologous recombination and chromosomal damage. The ability to track the normally elusive homologous recombination at stalled replication forks, afforded by DNA2 degradation, will allow us to define novel mediators of the pathway. These mechanistic insights will shed important new light on how cells deal with obstacles to DNA replication and maintain genome stability while proliferating. Our findings have the potential to identify new therapeutic targets for the treatment of cancers that are highly dependent on proper replication fork recovery and will help explain the growth defects in Seckel syndrome patients.
One prominent pathway of replication fork recovery is homologous recombination. This pathway is particularly important when the conventional replication machinery dissociates from the fork. Homologous recombination proteins can assemble a surrogate DNA synthesis machinery that promotes the continuation of replication. However, recombination-dependent DNA synthesis copies DNA less faithfully and can also lead to mutations and chromosomal changes. It is currently not clear how cells decide when to deploy homologous recombination at stalled replication forks and how they balance the risks and benefits associated with this pathway. It seems likely that cells only use this pathway when necessary and preferentially use mechanisms that directly restart the fork retaining the conventional replication machinery, hence avoiding the risk of mutations. One factor implicated in this direct form of fork restart is DNA2. Highlighting its biological significance, cells are unable to survive without DNA2 and its dysfunction results in a congenital dwarfism disorder known as Seckel syndrome. Elevated DNA2 levels on the other hand have been observed in cancer and it is thought DNA2 helps cancer cells to survive frequent replication fork stalling. We find that experimental degradation of DNA2 results in unusually high levels of homologous recombination DNA synthesis at stalled replication forks and cells enter a permanently non-dividing state. This not only explains why DNA2 is essential, but also provides a unique opportunity to determine how other fork restart pathways work alongside DNA2, and how the complex network of restart factors cooperates to choose the most appropriate mechanism to restart forks. Thus, our analysis will elucidate how extensively cells deploy homologous recombination at stalled replication forks and which regions of the genome are most vulnerable to unwarranted homologous recombination and chromosomal damage. The ability to track the normally elusive homologous recombination at stalled replication forks, afforded by DNA2 degradation, will allow us to define novel mediators of the pathway. These mechanistic insights will shed important new light on how cells deal with obstacles to DNA replication and maintain genome stability while proliferating. Our findings have the potential to identify new therapeutic targets for the treatment of cancers that are highly dependent on proper replication fork recovery and will help explain the growth defects in Seckel syndrome patients.
Technical Summary
DNA replication is impeded by obstacles and stresses that result in RF stalling. Central to RF recovery is RF reversal. Reversed RFs can either revert into a canonical RF and resume replication, or undergo homologous recombination (HR), whereby DNA synthesis is recovered in the context of a displacement-loop. HR-dependent DNA synthesis can facilitate replication completion but is error-prone and risks chromosome rearrangements. How cells balance RF restart with HR-dependent DNA synthesis for appropriate recovery pathway choice remains poorly understood.
Our unpublished results reveal that acute degradation of Seckel-syndrome helicase-nuclease DNA2 invariably results in substantial HR-dependent DNA synthesis downstream of RF stalling and is accompanied by immediate exit from the cell cycle. Previously, DNA2 has been implicated in the processing of reversed RFs, thereby mediating direct restart. Thus, RFs which escape the attention of DNA2 undergo opportunistic HR, which allows us to track, by immunofluorescence microscopy, large accumulations of HR-dependent RPA-ssDNA at endogenously stalled RFs marked by FANCD2. Using this unique system, we will screen for modifiers of HR-dependent DNA synthesis and probe the network of fork remodellers and restart pathways to determine how they impinge on the levels of HR at stalled RFs. How commonly fork restart factors act to supress HR and how this affects global replication will be delineated from DNA fibre assays. By ChIP-Seq, we will reveal genomic sites of HR-dependent DNA synthesis and explore whether commonly observed chromosome aberrations might be driven by this process. Our results will provide unique insight into how cells deal with endogenously stalled RFs and maintain genome stability while proliferating. The findings could identify new therapeutic targets for the treatment of cancers that are highly dependent on proper replication fork recovery and will help explain the growth defects in Seckel syndrome patients.
Our unpublished results reveal that acute degradation of Seckel-syndrome helicase-nuclease DNA2 invariably results in substantial HR-dependent DNA synthesis downstream of RF stalling and is accompanied by immediate exit from the cell cycle. Previously, DNA2 has been implicated in the processing of reversed RFs, thereby mediating direct restart. Thus, RFs which escape the attention of DNA2 undergo opportunistic HR, which allows us to track, by immunofluorescence microscopy, large accumulations of HR-dependent RPA-ssDNA at endogenously stalled RFs marked by FANCD2. Using this unique system, we will screen for modifiers of HR-dependent DNA synthesis and probe the network of fork remodellers and restart pathways to determine how they impinge on the levels of HR at stalled RFs. How commonly fork restart factors act to supress HR and how this affects global replication will be delineated from DNA fibre assays. By ChIP-Seq, we will reveal genomic sites of HR-dependent DNA synthesis and explore whether commonly observed chromosome aberrations might be driven by this process. Our results will provide unique insight into how cells deal with endogenously stalled RFs and maintain genome stability while proliferating. The findings could identify new therapeutic targets for the treatment of cancers that are highly dependent on proper replication fork recovery and will help explain the growth defects in Seckel syndrome patients.
