Final Report Summary - SLX5-8 IN DNA REPAIR (Ubiquitination and sumoylation in modulating subnuclear localization of DNA repair)
Replication fork stability has a critical role in human health. This project aimed to investigate proteins and mechanisms that affect replication fork stability or fork-related damage / repair. Our study revealed that POL32 and SLX5/SLX8 do not function in the same pathway with regard to HU- and MMS-sensitivity and that Pol32 has an important role at stalled and collapsed replication forks. Pol32 is the third subunit of Pol delta which consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We had constructed numerous pol31 alleles by alanine substitution for conserved amino acids and isolated six novel temperature- / cold-sensitive mutants of pol31. The ts and specifically cs mutants also show HU- and MMS-sensitivity. Combining the library of pol31 alleles with pol32 mutant showed that almost all pol31 mutations confer lethality in the absence of Pol32. Intriguingly, three temperature-permissive alleles of pol31 have been identified as suppressors of the phenotypic characteristics of pol32 null mutant i.e. suppressors of the cold-, HU- and MMS-sensitivities. These pol31 alleles and pol32 null mutant provided the required tool to investigate resumption of replication and/or polymerase stability when replication fork stalls on HU or collapses on HU+MMS.
Recovery assays showed that the ts and especially cs mutants of pol31 impair resumption of replication after replication fork stalling (HU). pol32 null mutant also shows similar phenotype and the suppressor mutants partially compensate for this pol32 defect. Our ChIP analyses showed that the absence of Pol32 destabilises Pol alpha and Pol epsilon at stalled replication forks and that the suppressor partially restores Pol epsilon de-stabilisation seen in pol32. De-stabilisation of Pol epsilon at stalled replication fork was also seen in the cold-sensitive mutant. The polymerase instability is not due to lack of checkpoint activation as Rad53 (CHK2) phosphorylation after HU treatment is intact in pol32 single and double mutant with its suppressor as well as in pol31 cs mutant. Intriguingly, cold temperature alone can trigger checkpoint activation in pol32 and pol31 cs mutants.
The non-catalytic subunits of Pol delta show more important role when cells are exposed to HU+MMS, which is thought to trigger fork collapse. ts/cs pol31 mutants and pol32 mutant significantly impair recovery after HU+MMS treatment, which is already reduced in wild-type cells. The suppressor mutants partially compensate for this pol32 defect. ChIP analyses in HU+MMS condition showed that there is no Pol epsilon enrichment at early firing origins, consistent with collapsed replication forks.
Recently, the crystal structure of the human orthologue of Pol31-Pol32N-term has been resolved. We took advantage of this structure to build homology model of Pol31. We mapped the positions of the mutations on it and found that the pol31 ts mutants probably alter the secondary structure of Pol31. The three suppressor alleles do not come together on the 3D structure, but the cs mutant and one of the suppressor mutations map to a solvent exposed loop on the surface of Pol31. Importantly, this surface patch has the highest conservation in Pol31 and is likely to be involved in protein-protein interactions. A potential partner for this conserved patch on Pol31, which is away from Pol32, can be the Pol3 C-terminal domain (CTD). We have explored this possibility by studying the interaction between Pol3-CTD and Pol31 in the cs and the suppressor pol31 mutants compare to WT-POL31 by yeast two-hybrid assay. Interestingly, the cs mutant results in the loss of interaction between Pol31 and Pol3-CTD while the suppressor mutant results in the enhancement of this interaction. These results are consistent with the phenotypes that these mutants confer and show the impact of Pol3-Pol31 interaction on the stability and function of DNA polymerases.
Recovery assays showed that the ts and especially cs mutants of pol31 impair resumption of replication after replication fork stalling (HU). pol32 null mutant also shows similar phenotype and the suppressor mutants partially compensate for this pol32 defect. Our ChIP analyses showed that the absence of Pol32 destabilises Pol alpha and Pol epsilon at stalled replication forks and that the suppressor partially restores Pol epsilon de-stabilisation seen in pol32. De-stabilisation of Pol epsilon at stalled replication fork was also seen in the cold-sensitive mutant. The polymerase instability is not due to lack of checkpoint activation as Rad53 (CHK2) phosphorylation after HU treatment is intact in pol32 single and double mutant with its suppressor as well as in pol31 cs mutant. Intriguingly, cold temperature alone can trigger checkpoint activation in pol32 and pol31 cs mutants.
The non-catalytic subunits of Pol delta show more important role when cells are exposed to HU+MMS, which is thought to trigger fork collapse. ts/cs pol31 mutants and pol32 mutant significantly impair recovery after HU+MMS treatment, which is already reduced in wild-type cells. The suppressor mutants partially compensate for this pol32 defect. ChIP analyses in HU+MMS condition showed that there is no Pol epsilon enrichment at early firing origins, consistent with collapsed replication forks.
Recently, the crystal structure of the human orthologue of Pol31-Pol32N-term has been resolved. We took advantage of this structure to build homology model of Pol31. We mapped the positions of the mutations on it and found that the pol31 ts mutants probably alter the secondary structure of Pol31. The three suppressor alleles do not come together on the 3D structure, but the cs mutant and one of the suppressor mutations map to a solvent exposed loop on the surface of Pol31. Importantly, this surface patch has the highest conservation in Pol31 and is likely to be involved in protein-protein interactions. A potential partner for this conserved patch on Pol31, which is away from Pol32, can be the Pol3 C-terminal domain (CTD). We have explored this possibility by studying the interaction between Pol3-CTD and Pol31 in the cs and the suppressor pol31 mutants compare to WT-POL31 by yeast two-hybrid assay. Interestingly, the cs mutant results in the loss of interaction between Pol31 and Pol3-CTD while the suppressor mutant results in the enhancement of this interaction. These results are consistent with the phenotypes that these mutants confer and show the impact of Pol3-Pol31 interaction on the stability and function of DNA polymerases.