Mrc1 and Tof1 regulate DNA replication forks in different ways during normal S phase

Abstract

The Mrc1 and Tof1 proteins are conserved throughout evolution, and in budding yeast they are known to associate with the MCM helicase and regulate the progression of DNA replication forks. Previous work has shown that Mrc1 is important for the activation of checkpoint kinases in responses to defects in S phase, but both Mrc1 and Tof1 also regulate the normal process of chromosome replication. Here, we show that these two important factors control the normal progression of DNA replication forks in distinct ways. The rate of progression of DNA replication forks is greatly reduced in the absence of Mrc1 but much less affected by loss of Tof1. In contrast, Tof1 is critical for DNA replication forks to pause at diverse chromosomal sites where nonnucleosomal proteins bind very tightly to DNA, and this role is not shared with Mrc1.

Figure 1.

The progression of DNA replication forks on chromosomes 3 and 6 in control cells during normal S phase. The chromosomal origins ARS305, ARS608, and ARS609 were deleted in the strains used in this work. (A and B) Progression of the leftward fork from ARS306 to the left end of chromosome 3. (C and D) Progression of the rightward fork from ARS607 to the right end of chromosome 6. Semiconservative replication of each site causes the corresponding restriction fragment to move from the heavy-heavy peak (HH) in the cesium chloride gradient to the heavy-light (HL) peak. The asterisk marks a less-dense peak that occurs transiently at the origins as they are replicated. This peak can also be detected 10 kb to the left of ARS306, but it is not seen further away from the origins, perhaps due to loss of synchrony as forks progress away from the site of initiation. We do not know at present whether the less-dense peak represents replication intermediates themselves (bubbles and forks) that are less dense than unbranched double-stranded DNA due to their structure, or whether it is produced by the annealing of the newly synthesized “light” daughter strands at replication forks.

Figure 2.

DNA replication forks progress very slowly in the absence of Mrc1. The layout and annotation are the same as in Figure 1.

Figure 3.

The rate of progression of DNA replication forks in the absence of Tof1 is more similar to control cells than to cells lacking Mrc1. The layout and annotation are the same as in Figure 1.

Figure 4.

Tof1 but not Mrc1 is required for DNA replication forks to pause at chromosomal sites that correspond to tRNAs. (A) The indicated strains were released from G1 arrest at 24°C in YPD medium, and samples were taken every 15 min. DNA content was measured by flow cytometry. (B and C) Two-dimensional DNA gels were used to detect replication intermediates at sites on chromosome 6 that correspond to the tRNAs tP(UGG)F and tA(AGC)F.

Figure 5.

The pausing of DNA replication forks at centromeres requires Tof1 but not Mrc1. (A and B) Two-dimensional DNA gels were used to detect replication intermediates at the centromeres of chromosomes 3 and 4.