New Insights into the roles of DNA replication/repair proteins at telomeres

Shilpa Sampathi

Efficient and complete replication of chromosomes is critical to prevent aberrant telomeres as well as to avoid unnecessary loss of telomere DNA. Due to the complexity in their structures and sequences, telomeres pose quite a challenge to the replication machinery in completely replicating the chromosomes. Telomeres end in a G rich 3' overhang which is extended by a cellular reverse transcriptase telomerase. It replenishes the telomeric repeats and maintains the functional telomeres. The complementary C strand is filled in by the replication machinery in coordination with telomere protection factors. In lower eukaryotes telomerase action is regulated by the replication factors, whereas these events remain elusive in higher eukaryotes. This dissertation aims at understanding the possible roles of DNA replication factors in telomere maintenance. It begins with a review of various replication/repair factors, telomeric proteins and their activities in conferring sound telomere replication. The first study assessed the role of flap endonuclease I (FEN1), a lagging strand replication protein, in telomerase mediated telomere maintenance. The findings revealed that FEN1 associates with catalytic component of telomerase, hTERT. FEN1 deficiency leads to shortened telomeres and nuclease defective mutant FEN1 showed telomere end to end fusions. The second study mapped the interaction domain of FEN1 with hTERT. The findings revealed that C terminal domain and the nuclease domain are involved for this association. Another study assesses Dna2, a lagging strand DNA exo/endonuclease and a helicase involved in RNA primer removal pathway. This study demonstrated that Dna2 localized to telomeres. Haploinsufficiency of Dna2 caused telomere fragility and increased damage response. Finally the dissertation sheds light on a pilot study involving PolA2, the regulatory B subunit of polymerase &alpha-primase complex. Pol &alpha-primase is speculated in priming the C strand synthesis with the aid of telomeric protection complexes. Our preliminary data revealed that PolA2 is needed for telomere maintenance, as depletion of PolA2 leads to increased G overhangs. Additionally PolA2 associates with Ten1 component of newly characterized CST complex, speculated to recruit Pol &alpha to telomeres. Taken together, the results presented here provide insights into the roles of replication factors for stable telomere maintenance.

New Insights into the roles of DNA replication/repair proteins at telomeres

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