Genetic and functional interactions between Mus81-Mms4 and Rad27

Abstract

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

“…An alternative fate of the 3Ј-flaps formed via flap equilibration is that they could be newly processed by a 3Ј-endonuclease such as Mus81 or its related complexes. This is in keeping with the observation that the overexpression of Mus81-Mms4 suppresses the lethality of dna2-K1080E (59).…”

The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing

“…The substitution of Asp with Ala at amino acid 179 in Rad27 was introduced by using a pair of mutagenic primers: 5Ј-GCC ACA CTC TGT TAT AGA AC-3Ј and 5Ј-CAT ATC TTC ACT TGC TGC GGC-3Ј (underlined at the mutagenic base). All truncated derivatives of Rad27 were cloned into pGEX-4T-1, and the resulting expression vectors produced Rad27 as a N-terminal GST fusion protein as described previously (34).…”

“…For this purpose, we used the N-terminal GST-tagged Rad27 derivatives (GST-Rad27 x-y ; where x and y indicate the amino acid residue in Rad27) that were used previously (34). We focused on the poorly conserved C-terminal region of Rad27, which is known to interact with many other proteins (34, 36, 40 -42).…”

A Saccharomyces cerevisiae RNase H2 Interaction Network Functions To Suppress Genome Instability

“…This is despite the fact that replication-generated Okazaki fragments, whose processing involves RNase H2, may be the largest source of DNA damage in the cell (29). Consequently, the obser-vation that mutations in RAD27 and DNA2 cause increased genome instability, including GCRs, potentially as a result of defects in Okazaki fragment processing (17,(33)(34)(35)(36)(37)(38)(39), suggests that defects in RNase H2 also cause increased genome instability due to defects in Okazaki fragment processing. Furthermore, ribonucleotides are misincorporated at significant levels by DNA polymerases during DNA replication (25).…”