The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. We show that exceptionally potent G-quadruplex unwinding is conserved amongst Pif1 helicases. Moreover, Pif1 helicases from organisms separated by >3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to novel genetic and epigenetic changes. Further, when expressed in yeast, human Pif1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.

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Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Background: An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae.

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.[Key Words: Chromosomal domains; chromosome walk; 2D agarose gels; Saccharomyces cerevisiae] Received January 17, 1996; revised version accepted May 28, 1996.The process of DNA replication is intricately controlled and integrated within the eukaryotic cell cycle. For ex ample, the onset of DNA replication is delayed in re sponse to damage of the DNA template (Siede et al. 1993), mitosis does not ensue until replication has been completed (Weinert andHartwell 1988, 1989), and rep lication of the genome is restricted to a single round during each S phase (Blow and Laskey 1988). Additional controls are exerted over the time at which chromosom al regions initiate replication. This temporal pattern can be visualized in mammalian cells by incorporating BrdU into DNA at discrete times during S phase and examin ing the chromosomes at mitois (e.g., Drouin et al. 1990). BrdU incorporation reveals interspersed early and latereplicating regions. The average DNA content of these regions is estimated at 2500-3000 kb, an amount suffi cient to contain 10-50 origins of replication (Drouin et al. 1990). Because most neighboring origins have been found by DNA fiber autoradiography to initiate replica tion fairly synchronously (Hand 1975), the origins loThese authors contributed equally to this work.