Saccharomyces cerevisiae RNase H(35) Functions in RNA Primer Removal during Lagging-Strand DNA Synthesis, Most Efficiently in Cooperation with Rad27 Nuclease

Abstract

Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.Replication of double-stranded DNA is an asymmetric process. While leading-strand synthesis proceeds continuously, lagging-strand synthesis takes place by synthesis, processing, and ligation of Okazaki fragments (39). These fragments, measuring about 200 nucleotides (nt) in eukaryotes, are primed by DNA polymerase alpha/primase with a short oligoribonucleotide of 7 to 14 residues. Before the nascent Okazaki fragments are ligated to form a continuous lagging strand, the short RNA primers must be removed by an enzyme exhibiting RNase H activity. Although several such enzymes from eukaryotes are known, the process of RNA primer hydrolysis is as yet not fully understood.An RNase H was first detected in calf thymus extracts (57). Subsequently, RNase H enzymatic activity was detected in all prokaryotes and eukaryotes examined as well as in a bacteriophage and in retroviruses as a part of reverse transcriptases (for reviews, see references 12 and 66). Generally, RNases H are defined as ribonucleotide-specific endonucleases, cleaving the RNA portion of RNA-DNA/DNA or RNA/DNA duplexes. Several RNases H implicated in RNA primer removal have been purified and/or cloned from diverse organisms ranging from bacteriophages to human cells (see, e.g., references 8, 9, 13, 14, 24, 26, 37, 51, and 52). Nevertheless, conclusive evidence of the involvement of these enzymes in primer removal is still lacking. In the budding yeast, Saccharomyces cerevisiae, three different RNases H were identified and partially characterized as RNase H(70), RNase H1, and RNase H(35) (17,32,34). These enzymes are evolutionarily related to prokaryotic as well as to mammalia...

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

“…35 and references therein), wherein "difficult to extend" termini can rearrange to misaligned intermediates with normal, correct terminal base pairs that, upon further extension, yield insertion/deletion mutations. Consistent with this possibility are studies (36,37) demonstrating that a yeast rnh35 strain has a spontaneous mutator phenotype. In those studies, the mutator effect was suggested to result from defective processing of RNA primers at the 5′-ends of Okazaki fragments.…”

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

“…Because Rad27 cleaves 5' flaps during Okazaki fragment processing, rad27Δ strains grow slowly [26] and are exquisitely sensitive to the yeast transformation procedure (data not shown). Overexpression of RNase H (RNH35), which catalyzes an alternate pathway of Okazaki fragment processing, suppresses the rad27Δ growth defects [27] and also reduces the toxicity of transformation (data not shown) without affecting the ratio of processed to simple religation NHEJ (see Figure 4B below). We therefore added an RNH35 overexpression plasmid to our strains.…”

Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts

“…In previous studies related to the removal of Okazaki primers (22,23), the human enzyme FEN-1 was found to be able to remove a single ribonucleotide at the 5Ј end of DNA. This makes FEN-1 a candidate enzyme for the second cut that we observe in substrates with a single ribose.…”