Anatomy and Dynamics of DNA Replication Fork Movement in Yeast Telomeric Regions

Abstract

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y′ telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y′ elements were found to fire in early-mid-S phase, while ARSs at the terminal Y′ elements were confirmed to fire late. An unfired Y′ ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase. The pause at telomeric sequence TG_1-3 repeats was stronger at the terminal tract than at the internal TG_1-3 sequences located between tandem Y′ elements. We show that the telomeric replication fork pause associated with the terminal TG_1-3 tracts begins ∼100 bp upstream of the telomeric repeat tract sequence. Telomeric pause strength was dependent upon telomere length per se and did not require the presence of a variety of factors implicated in telomere metabolism and/or known to cause telomere shortening. The telomeric replication fork pause was specific to yeast telomeric sequence and was independent of the Sir and Rif proteins, major known components of yeast telomeric heterochromatin.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org.

We thank members of the Blackburn and Herskowitz labs for support and encouraging discussions. We acknowledge Jennifer Fung, Jue Lin, Tanya Williams, and anonymous referees for critical reading of the manuscript and helpful suggestions.

This work was supported by NIH grants GM59466 (to I.H.) and GM26259 (to E.H.B.)