Genomic instability contributes to many diseases, such as cancer, neurodegenerative disease, and developmental disorders, but it also underlies many natural processes including aging, evolution, and antibody diversification. The Cimprich lab is focused on understanding the mechanisms that the cell uses to maintain genomic stability, with an emphasis on the DNA damage response. This is a complex, multi-faceted response that requires cells to sense the presence of DNA damage within the vast genome, as well as to “choose” and coordinate a wide range of downstream events and outcomes. These include effects on DNA repair, transcription, and DNA replication, as well as arrest of cell cycle progression, apoptosis, and senescence.

We are particularly interested in understanding how DNA damage is identified and resolved during DNA replication. The genome is particularly vulnerable during DNA replication because the replisome stalls at many DNA lesions. Stalled replication forks are unstable and can be processed in aberrant ways, leading to double-strand break formation and chromosomal rearrangements. Thus, cells must rapidly stabilize stalled forks and initiate a pathway to restart and complete DNA replication.

The lab studies the DNA damage response using cultured mammalian cells, as well as cell-free extracts derived from the eggs of the frog Xenopus laevis. We use these systems and a range of multidisciplinary techniques to understand how the DNA damage response is initiated, how this pathway is integrated with the processes of DNA replication and transcription, and how cells recover from DNA damage.

Specific areas of current interest are:

Checkpoint Activation and Signaling. We are interested in understanding how the DNA damage response is initiated, and how it regulates DNA replication and DNA repair. As one focus, we are studying the nature of the DNA damage signal(s) sensed by the cell and the role that replication plays in its generation. We are also interested in understanding how cells coordinate DNA replication with the DNA damage response. In particular, we are interested in how stalled replication forks are stabilized, so as to prevent further DNA damage, and how replication resumes at a stalled replication fork. We have identified new factors involved in these processes and ongoing projects relate to studying the roles of these new factors and other known DNA damage response proteins in these processes.

DNA Damage Tolerance. DNA damage tolerance (DDT) pathways (also known as post-replication repair pathways) promote the completion of replication when a fork stalls at a lesion by providing a mechanism to fill in gaps created by replication barriers while putting off repair of the lesion to a more convenient time. One form of DDT involves direct lesion bypass by translesion synthesis (TLS), and involves the use of specialized polymerases. The other form of DDT involves a template switch, in which the sister chromatid serves as a template for indirect bypass of the lesion. Although DDT pathways help to promote continued replication, thereby suppressing prolonged fork stalling and fork collapse, direct lesion bypass can be mutagenic. We are interested in understanding the mechanisms involved in DDT, and how the cell balances mutagenic and non-mutagenic DDT pathways.

New Pathways for Genome Stability: Recently we performed a genome-wide siRNA screen to define the processes and proteins that protect cells from DNA damage, particularly during DNA replication. This screen identified many known DNA damage response proteins as well as numerous unknown proteins. We are currently following up on several proteins from this screen, and are carrying out additional secondary screens as well to narrow our focus to those hits relevant to replication fork stabilization and to identify other new genome maintenance factors.

Transcription, RNA, and DNA Damage. Results from the genome-wide screen described above revealed that RNA is another prominent and unexplored source of genome instability and DNA damage in the cells. Specifically, we found that defects in some RNA processing genes lead to increased DNA damage through the formation of toxic RNA-DNA hybrids and R-loops. We are interested in studying how these structures arise, how they lead to DNA damage, and the nature of their physiological function. We are also interested in understanding how cells deal with transcription complexes during DNA replication, as these can act as replication fork barriers and promote fork arrest.

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