DNA and Chromosomes
Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis
Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ.The telomere-binding protein Rif2 and ATP-bound Rad50 have opposing roles in the activation of yeast Tel1ATM kinase
Saccharomyces cerevisiae Tel1 is the ortholog of human ATM kinase and initiates a cell cycle checkpoint in response to dsDNA breaks (DSBs). Tel1ATM kinase is activated synergistically by naked dsDNA and the Mre11-Rad50-Xrs2NBS1 complex (MRX). A multisubunit protein complex, which is related to human shelterin, protects telomeres from being recognized as DSBs, thereby preventing a Tel1ATM checkpoint response. However, at very short telomeres, Tel1ATM can be recruited and activated by the MRX complex, resulting in telomere elongation.Activation of Tel1ATM kinase requires Rad50 ATPase and long nucleosome-free DNA but no DNA ends
In Saccharomyces cerevisiae, Tel1 protein kinase, the ortholog of human ataxia telangiectasia–mutated (ATM), is activated in response to DNA double-strand breaks. Biochemical studies with human ATM and genetic studies in yeast suggest that recruitment and activation of Tel1ATM depends on the heterotrimeric MRXMRN complex, composed of Mre11, Rad50, and Xrs2 (human Nbs1). However, the mechanism of activation of Tel1 by MRX remains unclear, as does the role of effector DNA. Here we demonstrate that dsDNA and MRX activate Tel1 synergistically.
