1. Volume 56, Issue 4, Pages 551-563 (November 2014)
Strand-Specific Analysis Shows Protein Binding at Replication Forks and PCNA Unloading from Lagging Strands when Forks Stall 
Chuanhe Yu, Haiyun Gan, Junhong Han, Zhi-Xiong Zhou, Shaodong Jia, Andrei Chabes, Gianrico Farrugia, Tamas Ordog, Zhiguo Zhang 
Molecular Cell 
Volume 56, Issue 4, Pages (November 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 551-563DOI: (10.1016/j.molcel.2014.09.017)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Polε and Polδ Bind Leading and Lagging Strands of HU-Stalled DNA Replication Forks, Respectively
(A) An outline of the experimental strategy for eSPAN method. Early S phase cells released from the G1 block in the presence of BrdU with/without hydroxyurea (HU) were used to perform BrdU immunoprecipitation (BrdU-IP) and protein chromatin immunoprecipitation (ChIP) of targeted protein. Protein-associated nascent single-stranded (ss) DNA was enriched with BrdU-IP. The isolated ssDNA from BrdU IP (BrdU IP-ssSeq), eSPAN, and protein ChIP (ChIP-ssSeq) was subjected to strand-specific sequencing. The sequence reads were mapped to both the Watson strand (W, red) and the Crick strand (C, green) of the reference genome.
(B) A snapshot of BrdU IP-ssSeq, protein ChIP-ssSeq and eSPAN peaks at ARS815 for Polε catalytic subunit (Polε), Polδ catalytic subunit (Polδ), and Pol32 (a subunit of Polδ subunit). The signals represent normalized sequence read densities. Red and green colors represent the Watson and Crick strand, respectively.
(C and D) Polε and Polδ associate with leading and lagging strands of replication forks, respectively. The average log2 ratios of sequence reads from Watson strands and Crick strands at 134 group I origins were calculated using a 200 bp window to obtain the average bias pattern after normalization against the corresponding BrdU-IP-ssSeq. The formula to calculate strand bias of eSPAN peaks at individual origins was shown at the top panel of (D) and was described in Experimental Procedures. Using this method, the eSPAN peaks at each of 134 origins were separated into three categories: leading-strand bias, indeterminable bias, and lagging-strand bias. The dot and box plot shows the variation of the ratio of sequence reads at lagging over leading strands of individual eSPAN peaks. The lagging/leading ratio was normalized against corresponding sequence reads of BrdU-IP. Each dot represents one eSPAN peak.
See also Figures S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Polε and Polδ Are Enriched at Leading and Lagging Strands of All Active Replication Forks, Respectively
(A) Fluorescence-activated cell sorting analysis of cell cycle progression of yeast cells released from G1 block using alpha factor into S phase without HU at 16°C.
(B and C) The eSPAN peaks of the Polε catalytic subunit exhibit a leading-strand bias pattern at both group I (B) and group II origins (C) during normal cell cycle progression (72, 84, and 96 min after release).
(D and E) The eSPAN peaks of the Polδ catalytic subunit exhibit a lagging-strand bias pattern at both group I (D) and group II (E) origins during normal cell cycle progression. The average bias of eSPAN peaks at group I and group II origins in (B)–(E) was analyzed as described in Figure 1C.
(F) Polε and Polδ eSPAN peaks exhibit leading- and lagging-strand bias at almost all individual group I origins. The eSPAN data using cells 72 min after release were used for the analysis as described in Figure 1D.
(G) A model for the association of Polε and Polδ with replication forks.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Analysis of Seven Proteins at HU-Stalled Replication Forks Using eSPAN
(A) Cdc45, Mcm6, and Mcm10 are enriched at leading strands of HU-stalled replication forks.
(B) A model explaining Cdc45 and Mcm6 data in (A).
(C) Polα, Rfa1, and Rfc1 bind preferentially to lagging strands of HU-stalled replication forks.
(D) A model explaining Polα in (C).
(E) Analysis of the strand-bias pattern of eSPAN peaks of six proteins in (A) and (B) at individual origins as described in Figure 1D.
(F) An example of PCNA eSPAN peaks at origins ARS606 and ARS607 for HU-stalled forks (+HU) and active forks (−HU).
(G) Analysis of the average bias pattern of the PCNA eSPAN peaks at HU-stalled replication forks as well as active forks.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
PCNA Unloading Depends on Elg1 and Partially on PCNA Ubiquitylation
(A) Box plot of relative amount of PCNA on leading and lagging strands with or without HU of group I origins. The PCNA relative amount is estimated by log2 ratio of sequence reads of PCNA eSPAN over PCNA protein ChIP.
(B) Representative PCNA eSPAN peaks at the origin ARS737 for wild-type (WT) and relevant mutations indicated on the left. Red and green colors represent the normalized read densities of the Watson and Crick strand, respectively.
(C) Average bias pattern of PCNA eSPAN peaks at group I origins in the HU-treated cells with relevant genome type shown on the right.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
PCNA Is Unloaded from Lagging Strands of MMS-Stalled DNA Replication Forks
(A) Two representative BrdU IP-ssSeq, PCNA ChIP-ssSeq, and PCNA eSPAN peaks at the ARS737 origin in WT and elg1Δ cells.
(B–E) Average bias profiles of PCNA eSPAN peaks in WT (B and C) and elg1Δ strains (D and E). Yeast cells arrested at G1 using alpha factor were released into S phase with 0.035% MMS. Cells were collected at 30 min (B and D) and 40 min (C and E) for eSPAN, BrdU IP-ssSeq, and PCNA protein ChIP-ssSeq.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
The Checkpoint Kinases Mec1 and Rad53 Regulate PCNA Unloading from Lagging Strands of HU-Stalled Replication Forks
(A and B) PCNA eSPAN peaks exhibit the lagging-strand pattern at HU-stalled forks in mec1Δsml1Δ, rad53Δsml1Δ, and rad53-1 mutant cells in contrast to the leading-strand bias pattern in wild-type cells. Average bias pattern of eSPAN peaks at group I origins was shown.
(C and D) Elg1 physically interacts with Rad53. Elg1 (C) or Rad53 (D) was immunoprecipitated (IP) from cells with or without HU treatment. Proteins in whole-cell extract (WCE) and IP were detected by western blotting using antibodies against the indicated proteins.
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Cells Deficient in PCNA Unloading Exhibit Genome Instability
(A and B) Uncontrolled DNA synthesis under replication stress was observed in cells deficient in PCNA unloading. (A) An example of BrdU IP-ssSeq peaks at ARS607 in WT and mutant cells with relevant mutations shown on the right. (B) Box plot showing the distribution of BrdU peak length in the same strains as in (A). The mean and SD of the BrdU peak length are indicated in kb. Asterisks indicate p < (two-tailed Student’s t test).
(C) BrdU-IP qPCR analysis confirms that DNA synthesis at ARS607 in the presence of HU continues further in elg1Δ and PCNA K164R mutant cells compared with wild-type cells. Cells arrested with alpha factor were released into fresh YPD with HU and BrdU for 45 min. DNA obtained from BrdU IP was analyzed by real-time PCR using primer pairs indicated in (A). Data represent mean ± SD from one BrdU-IP experiment. Similar results were obtained in an independent experiment.
(D) Cells deficient in PCNA unloading exhibit increased chromosome breaks. The percentage of cells with Rad52 foci with/without HU treatment was reported. Data represent mean ± SEM.
(E) Model summarizing the effect of different mutations on PCNA unloading from HU-stalled forks. This model was based on the idea that the ratio of PCNA trimers on lagging stranding over leading strands at normal forks is 2:1.
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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