Y RNA functions at the initiation step of mammalian chromosomal DNA replication

Torsten Krude,Kathrin Marheineke,Olivier Hyrien,Christo P Christov

doi:10.1242/jcs.047563

Abstract

Non-coding Y RNAs have recently been identified as essential novel factors for chromosomal DNA replication in mammalian cell nuclei, but mechanistic details of their function have not been defined. Here, we identify the execution point for Y RNA function during chromosomal DNA replication in a mammalian cell-free system. We determined the effect of degradation of Y3 RNA on replication origin activation and on fork progression rates at single-molecule resolution by DNA combing and nascent-strand analysis. Degradation of Y3 RNA inhibits the establishment of new DNA replication forks at the G1- to S-phase transition and during S phase. This inhibition is negated by addition of exogenous Y1 RNA. By contrast, progression rates of DNA replication forks are not affected by degradation of Y3 RNA or supplementation with exogenous Y1 RNA. These data indicate that Y RNAs are required for the establishment, but not for the elongation, of chromosomal DNA replication forks in mammalian cell nuclei. We conclude that the execution point for non-coding Y RNA function is the activation of chromosomal DNA replication origins.

Highlights

Replication of chromosomal DNA in eukaryotes is regulated predominantly at the level of origin activation, which leads to the establishment of DNA replication forks during S phase of the celldivision cycle
Origin activation depends on the cyclin- and Dbf4-dependent protein kinase activities of Cdk2 and Cdc7, respectively, which convert pre-replicative complexes (pre-RCs) into active DNA replication forks via the recruitment of DNA polymerases and additional replication factors
In fractionation and reconstitution experiments using human cell extracts, we have recently identified the small non-coding Y RNAs as novel factors that are essential for chromosomal DNA replication in isolated G1-phase nuclei (Christov et al, 2006)

Summary

Introduction

Replication of chromosomal DNA in eukaryotes is regulated predominantly at the level of origin activation, which leads to the establishment of DNA replication forks during S phase of the celldivision cycle (reviewed by Costa and Blow, 2007; Gilbert, 2001; Machida et al, 2005). Origin activation depends on the cyclin- and Dbf4-dependent protein kinase activities of Cdk and Cdc, respectively, which convert pre-RCs into active DNA replication forks via the recruitment of DNA polymerases and additional replication factors This regulation network is evolutionarily conserved in eukaryotes from yeast to human; additional levels of control have evolved in metazoan organisms (reviewed by Arias and Walter, 2007). In fractionation and reconstitution experiments using human cell extracts, we have recently identified the small non-coding Y RNAs as novel factors that are essential for chromosomal DNA replication in isolated G1-phase nuclei (Christov et al, 2006)

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