Biology subjects

Blow, J. J.

Publications and source records attributed to Blow, J. J..

4 recordsLinked to original sources

bioRxiv2023

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

molecular biology↗

bioRxiv2021

High levels of origin licensing during Xenopus cleavage divisions ensures complete and timely genome duplication

Cells face several challenges to completing genome duplication. One challenge is the irreversible stalling of converging replication forks ( double fork stalls). Cell types that cannot delay mitotic entry must also ensure that no replication origins are too far apart (the random gap problem). We show how these challenges can be met in early Xenopus embryos by the very abundant licensing of replication origins: one MCM2-7 double hexamer every [~]250 bp. Licensing does not change nucleosome spacing, consistent with MCM2-7 being assembled onto inter-nucleosomal linker DNA. We show that later embryonic development can occur successfully with a per-cell cycle failure rate of <0.2% in early embryos. The high density of licensed origins in the early embryo reduces cell cycle failures from random gaps and from double fork stalls to levels compatible with subsequent development, suggesting that Xenopus early embryonic cells can ensure complete genome duplication without requiring unconventional replication mechanisms.

biochemistry↗

bioRxiv2018

A histone H4K20 methylation-mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. The mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here, we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, appropriate chromatin relaxation is safeguarded by SET8-dependent methylation of histone H4 on lysine 20. Thus, in the absence of either SET8 or the H4K20 residue, substantial genome-wide chromatin decompaction occurs which allows excessive loading of the Origin Recognition Complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and the loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

molecular biology↗

bioRxiv2017

Delayed activation of the DNA replication licensing system in Lgr5(+) intestinal stem cells

During late mitosis and early G1, replication origins are licensed for replication by binding to double hexamers of MCM2-7. Here, we investigate how licensing and proliferative commitment are coupled in the small-intestinal epithelium. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5(+) stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem-cells is caused by an increased G1 length, characterised by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost In Apc mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G1 entry. We propose that the unlicensed G1 of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.

cell biology↗