Daochun Kong, Ph.D.
Control of cell division cycle
Professor, College of Life Sciences, Peking University
tel：
E-mail：kongdc@pku.edu.cn
1.The mechanisms of chromosomal DNA replication initiation, selection of replication initiation sites, and biochemical reactions in replication forks
2.The mechanism of checkpoint-regulated replication fork stability
3.The repair of double-stranded DNA breaks
The mechanism of DNA replication going through nucleosomes
How DNA replication forks go through nucleosomes is a long-standing question since 1990s. Our recent work shows that eukaryotic cells may use a series of histone modifications to relax nucleosomes for replication forks to move through nucleosomes. We have determined some histone modification sites and their chemical modifications critical for replication forks moving through nucleosomes.
The mechanism of arranging thin and long DNA strand in nucleus
How is a thin and long genomic DNA strand arranged to avoid being a random coil? We are working on this question and try to determine what proteins are involved in this biological event. Some protein candidates relevant to this event are being examined.
Instability or collapse of stalling DNA replication forks is thought as a greatest source of genomic instability during normal cell growth cycles. Checkpoint has been demonstrated as an essential cellular regulation for stabilization of stalling replication forks, but its fundamental mechanism of regulation remains to be determined. It is also unknown whether eukaryotic cells have other regulation pathways to stabilize stalling replication forks. For the above two questions, we have had important findings.
By inhibiting replicative helicase activity, the intra-S phase checkpoint prevents stalling replication forks from collapse
A vital challenge for DNA replication in eukaryotes is replication fork stalling and subsequent fork collapse. The intra-S phase checkpoint is essential for stabilization of stalled forks, but its central mechanism of regulation remains to be determined. In this study, we found that replication stress (fork stalling) results in a ~75% decrease in CMG helicase activity in wild-type but not in cds1 Chk2- cells in the fission yeast S. pombe. We demonstrated that the fork stalling-elicited reduction of CMG helicase activity results from the Cds1Chk2-mediated phosphorylation of Cdc45 subunit of CMG helicase on S275, S322 and S397 sites. Both phosphor-deficient (Cdc45-3A) and phosphor-mimic (Cdc45-3D) mutations on the three serine residues reduced CMG helicase activity by approximately 50% and rescued the sensitivity of cds1 Chk2- cells to hydroxyurea by approximately 15,000 folds. These results suggest that reducing the replicative helicase activity of CMG complex is a crucial step of checkpoint regulation in preventing stalling replication forks from collapse.
Replication fork stalling elicits chromatin compaction for stabilization of stalling forks
Replication forks stalling is a frequent event as replication forks traverse across chromatin DNA. Stalling replication forks require sophisticated cellular regulations to prevent their collapse. In this study, we found that fork stalling elicits chromatin compaction, and the chromatin compaction resulted from histone modification changes, including H2BK33 and H3K27 deacetylation and H3K9 tri-methylation. The mutation of H2BK33A/Q affected the formation of fork stalling-elicited chromatin compaction and thus significantly increased collapse of stalling forks. Furthermore, we demonstrated that H2BK33 deacetylation is independent of checkpoint regulation. Thus, fork stalling-elicited chromatin compaction is a distinct cellular regulation, and it functions in parallel with checkpoint regulation. We name this regulation as the “CCSSRF” (Chromatin Compaction Stabilizes Stalling Replication Forks) pathway.
Checkpoint regulates ubiquitin E3 ligase Brl2 to stabilize stalling replication forks
We discovered a new checkpoint target, the ubiquitin E3 ligase Brl2. When replication forks stalled, Brl2 was phosphorylated by checkpoint kinase Cds1Chk2 on five serine residues. Disruption of Cds1-mediated phosphorylation of Brl2 dramatically decreased the stability of stalling replication forks.
The protection mechanism of broken DNA ends
We demonstrated that broken DNA ends need to be protected to prevent genetic deletions. The mechanism of broken end protection is being examined.
H2BK78 acetylation is required for efficient repair of dsDNA breaks through homologous recombination
We found that H2BK78 is acetylated upon dsDNA breaks and the acetylation of H2BK78 is catalyzed by Gcn5. The disruption of H2BK78 acetylation (H2BK78R mutation) or Gcn5 defection remarkably affected the repair of dsDNA breaks. We further determined that H2BK78 acetylation facilitates end resection, a key step in the process to repair dsDNA breaks.
Zeng M, Tang ZZ, Ren LFD, Wang HB, Wang XJ, Zhu WY, Mao XB, Li ZY, Mo XM, Chen J, Han JH, Kong DC, Ji JG, Carr AM, Liu C. (2023) Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1. J. Clin. Invest., 133: e171533.
Sun YA, Xu X, Zhao WX, Zhang Y, Chen KY, Li YZ, Wang XT, Zhang ML, Xue BX, Yu WT, Hou YP, Wang CB, Xie W, Li C, Kong DC, Wang S, Sun YJ. (2023) RAD21 is the core subunit of the cohesin complex involved in directing genome organization. Genome Biol., 24: 155.
Liu SJ, Li XZ, Liu XQ, Wang JN, Li LY, Kong DC. (2022) RNA polymerase III directly participates in DNA homologous recombination. Trends Cell Biol., 32: 988-995.
Li XZ, Wang L, Liu XQ, Zheng ZQ, Kong DC. (2022) Cellular regulation and stability of DNA replication forks in eukaryotic cells. DNA Repair, 120: 103418.
Tang ZZ, Zeng M, Wang XJ, Guo C, Yue P, Zhang XH, Lou H Q, Chen J, Mu DZ, Kong DC, Carr AM, Liu C. (2022) Synthetic lethality between TP53 and ENDOD1. Nat Commun., 13: 2861.
Liu XQ, Chang FR, Liu SJ, Wu F, Kong DC. (2022) The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks. Yi Chuan, 44: 609-617.
Liu SJ, Hua Y, Wang JN, Li LY, Yuan JJ, Zhang B, Wang ZY, Ji JG, Kong DC. (2021) RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination. Cell, 184: 1314-1329.
Liu Y, Wang L, Xu X, Yuan Y, Zhang B, Li ZY, Xie YC, Yan R, Zheng ZQ, Ji JG, Murray JM, Carr AM, Kong DC. (2021) The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes. Proc. Natl. Acad. Sci. U. S. A., 118: e2019183118.
Liu SJ, Kong DC. (2021) A direct role of RNA polymerase III and RNA in DNA homologous recombination. Mol. Cell Oncol., 8: 1935173.
Liu SJ, Liu XQ, Kong DC. (2021) A Recently Discovered Essential Factor for DNA Homologous Recombination: RNA Polymerase III. Journal of Cell Science & Therapy, 12: 302.
Feng G, Yuan Y, Li ZY, Wang L, Zhang B, Luo JC, Ji JG, Kong DC. (2019) Replication fork stalling elicits chromatin compaction for the stability of stalling replication forks. Proc. Natl. Acad. Sci. U. S. A.,116: 14563-14572.
Jingna Wang, Yan Xia, Wenli Deng, Sijie Liu, Xin Xu, Rui Li, Bo Zhang, Zeqi Zheng, Feiran Chang, Jiechen Luo, Changru Yang, Ruishuang Meng, Fei Wu, Yu Chen, Yifan Jin, Jiawei Xie, Lingyan Li, Wotian Kuang, Zilu Wang
