Regulation of MDC1 during Mitotic DNA Damage Pubblico

Abstract

Genetic instability is a hallmark of human cancers. DNA double strand break (DSB), if improperly repaired, results in genetic instability and tumorigenesis. Previous research has established γH2AX as a bona fide marker of DSBs and that hierarchical foci assembly of DNA damage response (DDR) proteins at DSB sites is required for efficient repair. Recent findings from our group demonstrated that during either spontaneous or induced prolonged mitosis, cancer cells acquire DSBs, which lead to further chromosomal abnormality (1). Deciphering the mechanism of such DSB accumulation may provide new insights into the mechanism of genetic instability. Several recent studies suggest a partial DDR during mitosis, wherein the DSBs are marked by γH2AX, but repair only takes place after mitotic exit (2, 3). We hypothesize that the increase in DSBs during prolonged mitosis is due to the lack of efficient DDR. To this end, we focus on the protein Mediator of DNA Damage Checkpoint 1 (MDC1), an important mediator in DSB repair. MDC1 directly binds to γH2AX, serves as a platform to accumulate/retain downstream DDR proteins at DSB sites and concomitantly initiates cell cycle arrest through its multiple interaction domains. In this dissertation, I present evidence that demonstrates Cyclin-dependent kinase 1 (CDK1) inhibits MDC1- γH2AX interaction and provide new insights into mechanisms in response to DNA damage during mitosis. Additionally, my results suggest F-Box and WD Repeat Domain-Containing 7 (FBW7), the substrate recognition component of the E3 ligase complex SCF (complex of SKP1, CUL1 and F-box protein) as a negative regulator of MDC1 in human cancer cells.

Table of Contents

Table of Contents
Chapter I...2
Introduction and literature review...2

DNA damage response...2
DNA damage repair foci formation...2
Major DSB repair pathways...4
Cell cycle checkpoints...5
MDC1, the master organizer of DDR factors in IRIF...6

MDC1- mediated γH2AX foci formation...7

SDT repeat mediated MDC1-MRN interaction...10

PST repeat-mediated MDC1 -DNA-PK interaction...11
MDC1 - CHK2 interaction...12
The TQXF cluster-mediated chromatin modification...13
MDC1 in Mitotic progression...14
MDC1 degradation mechanism...14
Current view on mitotic DNA damage repair...15

Goal of this dissertation...16
Figure 1. Schematic illustration of IRIF foci formation...18
Figure 2. Cell cycle checkpoints...19
Figure 3. Spindle Assembly Checkpoint...20
Figure 4. MDC1, a hub in DSB repair...21

Chapter II...22
CDK1 Regulates Mediator of DNA Damage Checkpoint 1 (MDC1) During Mitotic DNA Damage...22

Abstract...23
Introduction...23
Materials and Methods...25
Results and Discussion...26
Figure 1. MDC1-γH2AX colocalization decreases during mitosis...33
Figure 2. MDC1 exhibits decreased interaction with γH2AX in mitosis...34
Figure 3. Inhibition of CDK1 activity increases MDC1-γH2AX colocalization in mitosis...35
Figure 4. CDK1 downregulation increases MDC1-γH2AX colocalization during mitosis...36
Supplemental Materials and Methods...37
Supplemental Figure S1. Mitotic indices of HCT116 cells during nocodazole treatment...39
Supplemental Figure S2. Enrichment of mitotic cells by mitotic shake-off...40
Supplemental Figure S3. Mitotic indices of cells...41
Supplemental Figure S4. Immunofluorescent staining of MDC1 and γH2AX in HCT116 cells...42
Supplemental Figure S6. siRNA knockdown of CDK1...44

Chapter III...45
FBW7 Regulates Mediator of DNA damage checkpoint 1...45

Abstract...46
Materials and Methods...49
Results...50
Discussion...53
Figure 1. PST repeat regulates MDC1 protein stability...55
Figure 2. FBW7 downregulates MDC1...58
Figure 3. Knockdown of FBW7 increases MDC1 level...60
Figure 4. Absence of FBW7 increases basal MDC1 level...61

Chapter IV...62
Summary and Future direction...62

Figure 1. Working model of MDC1- γH2AX interaction by CDK1 regulation...68

References...69

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular
Parola chiave	CDK1 mitosis MDC1
Committee Chair / Thesis Advisor	Yang, Vincent W, Emory University
Committee Members	Wilkinson, Keith D, Emory University Joshi, Harish C, Emory University Yao , Xuebiao , Morehouse School of Medicine Yun, Chris, Emory University Pallas, David C, Emory University

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