Lecture Notes

1 Introduction The instructors and students will introduce themselves, their backgrounds and reasons for participating in the course. Instructors will discuss the structure of the course, including requirements and assignments. The instructors will then present a short overview of cell-cycle regulation of DNA replication and the steps of DNA replication to give the students background information. Finally, we will briefly discuss the topic for next week.
2 The Cell Cycle For cells to properly divide, each stage of the cell cycle must be controlled so one stage does not begin before the completion of the previous stage. The two papers assigned for this week describe experiments that resulted in major breakthroughs in our understanding of how cellular division is controlled. The first paper, Evans et al., describes the identification of cyclins, which are now known to be specificity subunits of Cyclin-Dependent Kinases (Cdks). This paper was the first example of how proteolysis might be used to control the cell cycle. The second paper, Shirodkar et al., gives insight into how cyclins control the stages of the cell cycle by showing that cyclins interact with transcription factors in a stage-specific manner. In particular, this paper studies the transcription factor E2F, which interacts with the tumor-suppressor protein Rb.
3 G1 Phase and Initiation of DNA Replication Although the DNA in a cell is not actively replicated until S phase, the process of initiating replication is begun earlier in the cell cycle. During the G1 phase of the cell cycle, a pre-Replicative Complex (pre-RC) forms at multiple sites on each chromosome. These sites are known as origins. This complex is required to mark sites that are capable of initiating replication and for recruiting components of the replication machinery. The first of the two assigned papers, Donovan et al., describes experiments that show that proteins of the pre-RC assemble at origins in a specific order. The second paper, Mailand and Diffley, provides data for how the cell regulates the formation of pre-RCs by stabilizing one of the pre-RC components, Cdc6.
4 S Phase and Replication Fork Regression During the S phase of the cell cycle, replication is initiated at origins, which result in bi-directional replication forks. All origins do not initiate at the same time; rather there is a temporal program controlling when each origin initiates. The data in the paper by Dimitrova and Gilbert show that even though the temporal program is carried out during S phase, the temporal constraints are established during G1. In order for bi-directional forks to replicate DNA, a number of proteins are necessary to unwind the DNA for the polymerases to carry out replication. The second paper, Marinsek et al, provides insight into the function of a recently identified complex of proteins, GINS, at the replication fork.
5 Cell Cycle Control of DNA Replication Once the cell replicates the DNA, it is imperative that a second round of replication does not begin before the cell divides. Failure to prevent a second round of replication can result in genomic instability, which can lead to tumorigenesis. The first paper this week, Hayles et al., is one of the many papers that show that Cdk activity directed by cyclins (CDK activity) is important to inhibit DNA replication. The second paper, Nguyen et al., shows the mechanisms of how CDK activity can prevent a second round of DNA replication.
6 Checkpoints Each time a cell divides, not only does the genome need to be duplicated, but the genome must be free of damage. DNA damage can result in genomic instability and chromosomal rearrangements, both of which can lead to cancer. If damage occurs, a checkpoint is activated, resulting in a reversible cell-cycle arrest. During this arrest period, the cell can repair the DNA before continuing with the remainder of the cell cycle. The first paper this week, Kuerbitz et al., shows that p53 is required for the cell to activate a G1 checkpoint. This paper was one of the first articles that defined a physiological role for the tumor-suppressor protein p53. In the second paper, Katou et al. describe experiments that show that two proteins, Mrc1 and Tof1, are responsible for maintaining replication forks (i.e. ensuring that the forks do not collapse) during the repair.
7 DNA Repair This week, we will discuss mechanisms for how the cell repairs different types of damage. During replication, polymerases could encounter altered nucleotides, or lesions, that cause the replication fork to stall. Depending on the type of lesion, this stalled fork might activate a checkpoint to repair the damage. However, the lesion can be bypassed by switching the replicative polymerase for a low-fidelity polymerase. The mechanism for recruiting these other polymerases is studied in this week’s first paper by Bienko et al. Sometimes the damage is too great to simply be replicated over, such as a double-strand break (DSB). In the second paper, Spangolo et al. describe the structure of a complex of proteins necessary to repair DSBs and the implications for the mechanism of these proteins.
8 Midterm Assignments Field trip to the Oncology Department of Novartis.
9 Development and Endoreduplication Not only is DNA replication regulated each cell cycle, but it is also regulated differently during different developmental stages. In some organisms, specific cells purposely induce multiple rounds of DNA replication without an intervening mitosis, which results in multiple copies of the genome within a single cell. This process is called endoreduplication. Verkest et al. demonstrate that the cell controls the initiation of endoreduplication via CDK activity. Initiation of origins can be affected by local transcription activity, which can change throughout the development of an organism. The results in the second paper show that changes in the transcriptional program of the HoxB domain during development affect the site of initiation at a nearby origin.
10 Recombination and Meiosis Meiosis is the specialized cell cycle that produces four haploid cells from a single diploid cell. To accomplish this goal, the cell undergoes a single round of replication followed by two consecutive rounds of division. During these two divisions, chromosomes must segregate properly such that each haploid cell has the correct number and type of chromosomes. During chromosome segregation, genetic recombination occurs. This week we will discuss how the cell coordinates pre-meiotic replication with recombination and how chromosomes are monitored to ensure that they segregate properly.
11 DNA Replication and Cancer One hallmark of cancer is the uncontrolled division of cells. Thus, many of the genes that are mutated in cancer cells encode products that regulate the cell cycle. As we have discussed previously, cancer arises through DNA damage and often genomic instability, both of which can occur due to errors in DNA replication. Recent studies on the mechanisms that lead to cancer have shown that there is a link between control of DNA replication and the onset of cancer. The two papers assigned for this week examine the roles of pre-RC components and pre-RC formation in causing a cancerous state.
12 Targeting Replication Components as Chemotherapy As we discussed during the last class, regulation of DNA replication is often disrupted in cancerous cells. This disruption can lead to inappropriate DNA replication, which might lead to the amplification of oncogenes and the onset of cancer. It is becoming clear that one mechanism of the action of chemotherapeutic drugs is to target DNA replication. This week we will read two papers that attempt to understand the mechanism of two different anticancer drugs and how these mechanisms are related to DNA replication.
13 Viral Reprogramming of the Cell Cycle Viruses must infect host cells to reproduce, because viruses do not contain all the necessary components to propagate themselves. Once inside of the host cell, the virus co-opts much of the cellular machinery to ensure its survival. The papers this week examine how viruses can control a host cell by corrupting the cell cycle. The first paper this week, Yew and Berk, describes how one of the adenovirus proteins blocks p53 function, which can result in cancer. The second paper, Wiebusch et al., describes the way that human cytomegalovirus prevents host genome replication to ensure the replication of the viral genome.
14 Final Assignments Presented Each student will give a 10-12 minute oral presentation of a paper related to one of the topics we discussed during the semester. The student will be responsible for giving a brief statement of the relevant background, explaining key figures and tables, briefly discussing the conclusions of the paper and proposing subsequent experiments that could be done to answer questions raised in the paper. There will be time after each presentation for a short discussion, generated by questions from other students.