Mechanistic Understanding of the Role of TRF1 in Telomere Replication

<p>Mammalian telomeres are hard to replicate, and the shelterin subunit TRF1 is important for facilitating the replication of telomeric DNA. Deletion of TRF1 results in several phenotypes that are thought to be associated with telomere replication stress, including ATR signaling, fork stalling, sister telomere associations, and fragile telomeres. Fragile telomeres are structures that resemble common fragile sites (CFSs), but how they are formed is not known. TRF1 functions in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Deletion of BLM leads to lagging strand specific fragile telomere formation. In this thesis, I report that analogous to CFSs, fragile telomeres in BLM-deficient cells involve double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs are repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promotes fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competes with PARP1-, LIG3-, and XPF-dependent alternative non-homologous end joining (alt-NHEJ). Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs. Even though BLM loss can induce BIR-dependent telomere fragility on the lagging strand, it fails to explain all other phenotypes caused by TRF1 loss, including leading strand fragile telomeres, ATR signaling, fork stalling, and sister telomere associations. Even the frequency of lagging strand fragile telomeres observed in BLM-deleted MEFs is not comparable to that in TRF1-deleted cells. Therefore, it is likely that TRF1 has functions other than recruiting BLM to prevent replication stress at telomeres. In this thesis, I report two newly-identified functional modules in TRF1, one in the TRFH domain and the other one, surprisingly, in the Myb domain. Mutating each module leads to extensive telomere fragility and telomere replication stress. Interestingly, the general transcription factor IIH (TFIIH) complex is also important for facilitating telomere replication. Deleting components of the TFIIH complex causes extensive fragile telomere formation and telomere replication stress. Lastly, I have determined the nature of sister telomere associations caused by TRF1 loss. They are alt-NHEJ-mediated sister telomere fusions caused by the partial loss of TRF2, indirectly as a result of TRF1 deletion. Altogether, this work provides detailed mechanistic insights into the role of TRF1 during telomere replication and the consequences of the loss of TRF1 functions.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2022

Author dc:creator

• Yang, Zhe

Contributors dc:contributor

• Titia de Lange

• telomere replication
• TRF1
• fragile telomeres
• BLM helicase
• break-induced replication
• alt‑NHEJ
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/683

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1692