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Title: Investigating the genetic requirements for the PCNA interacting motifs in Pif1 family helicases in Saccharomyces cerevisiae
Author: Kotenko, Oleksii
ISNI:       0000 0004 9349 2410
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Stable maintenance and transmission of the genetic material requires tight coordination between DNA replication and DNA repair. One of the factors that couples DNA replication and DNA repair is proliferative cell nuclear antigen (PCNA). PCNA is a homotrimeric complex that encircles DNA and plays a structural role interacting with different enzymes, tethering them to DNA and promoting their catalytic activity. PCNA is heavily post-translationally regulated, which was shown to change its affinity to the interacting partners and therefore to confer differential recruitment of the required proteins to the DNA substrates. Pif1 family of DNA helicases in Saccharomyces cerevisiae is composed of the two paralogues Pif1 and Rrm3. In addition to the overlapping functions during DNA replication, Pif1 and Rrm3 have certain distinct roles. Rrm3 is required for DNA replication through the “hard-to-replicate” regions, such as rDNA repeats, telomeres and origins of replication. The unique functions of Pif1 involve telomerase inhibition at telomeres and at DNA double-strand breaks (DSBs) and promotion of break induced replication (BIR). The underlying mechanism that defines functional specialisation of Pif1 family helicases is not understood. Both Pif1 and Rrm3 were shown earlier to physically interact with PCNA. The experiment presented here shows that, in addition to the previously reported C-terminal PCNA interacting peptides (PIP1 and PIP2), the N-terminus of Pif1 contains putative PCNA interacting motifs (PIP3 and SIM). The Pif1 roles in DNA replication, BIR and telomerase inhibition require different PCNA-interacting motifs. In addition, Pif1 recruitment to stalled forks and DSBs was PCNAdependent. Despite the fact that the Pif1-PCNA complex was shown to be required for Pol δ-mediated DNA synthesis during BIR, it is still not clear if other stages of this DNA repair pathway are Pif1-dependent. This study shows that the broken strand invasion and the initiation of DNA synthesis during BIR occur independently of Pif1, however both the long-range DNA synthesis and re-synthesis of the resected strand require Pif1 and its previously reported Rad53/Dun1-dependent phosphorylation. Based on the published data, the interaction between Rrm3 and PCNA occurs via the N-terminally located PCNA interacting motif (PIP1 in Rrm3). The experiment presented here shows that PIP1 in Rrm3 was not required for DNA replication through the sites that require Rrm3 helicase. In contrast, replication associated function of Rrm3 required the C-terminally located putative PCNA interacting motif (PIP2 in Rrm3), homologous to the previously reported C-terminal PCNA interacting motif in Pif1 (PIP2 in Pif1). Overall, this work provides a systematic characterisation of the newly identified and previously reported pif1 and rrm3 alleles and shows that different functions of Pif1 can be distinguished by the genetic requirements for its PCNAinteracting elements. Based on the obtained data, I would like to hypothesise that differential recruitment of Pif1 helicases via interaction with PCNA could be the reason of the functional specialisation of Pif1 and Rrm3.
Supervisor: Makovets, Svetlana ; Buonomo, Sara Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: DNA double-strand breaks ; DSBs ; telomeres ; Pif1 family helicases ; Rrm3 ; PCNA interacting motifs