Spo11 generates gaps through concerted cuts at sites of topological stress.

Meiotic recombination is essential for chromosome segregation at meiosis and fertility. It is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homologue of an archaeal topoisomerase (Topo VIA). Here we describe previously uncharacterized Spo11-induced lesions, 34 to several hundred base pair-long gaps, which are generated by coordinated pairs of DSBs termed double DSBs.

Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms.

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb).

Chromatin Immunoprecipitation of Histone Modifications in Fission Yeast.

Chromatin immunoprecipitation (ChIP) is a sensitive, accurate, and reliable technique widely used to analyze protein-DNA interactions at specific binding sites in vivo. It has been a particularly powerful technique for mapping of histone modification patterns both at individual loci and genome-wide. Here we provide a detailed protocol for ChIP of histone modifications associated with active transcription in fission yeast (Schizosaccharomyces pombe).

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe.

The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes.