DNA repair pathway coordination during damage processing

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Project Summary/Abstract UV light, carcinogen exposure from air pollution and other environmental agents can damage DNA. The resulting base damages, if not properly processed and removed, can lead to DNA mutations, causing cell death or cellular transformation manifested in a wide variety of human maladies including aging, cancer, and neurodegenerative diseases. While canonically UV-damage and oxidative damage are repaired by nucleotide excision repair (NER) and base excision repair (BER), respectively, recent work indicates that key damage sensor proteins are not siloed within one repair pathway but work cooperatively in both BER and NER: these include poly(ADP-ribose) polymerase 1 (PARP1) (10-17), UV-damaged DNA binding protein (UV-DDB; a heterodimer of DDB1/DDB2) (18- 20), and xeroderma pigmentosum complementation group C protein (XPC-RAD23B). I hypothesize that key damage sensor proteins work together on multiple forms of damage to facilitate BER and NER, that handoff mechanisms depend on the type of DNA damage, and that handoff is essential to process damage embedded within nucleosomes. This hypothesis will be tested with single molecule and cell biology experiments with the completion of the following aims: AIM 1: To determine how DNA damage sensors cooperate at UV photoproducts. In subaim 1a, DNA binding positions, binding lifetimes, and mean squared displacement (for motile events that slide on the DNA) will be analyzed for UV-DDB, XPC-RAD23B, and PARP1 on UV photoproducts. Subaim 1b will investigate the kinetics of lesion hand-off for these three proteins by studying them in pairs (at equal concentrations) using orthogonal labels. In subaim 1c, the binding parameters of these three proteins will be assessed on nucleosomes with damaged DNA, with the proteins analyzed alone or with relevant nucleosome interacting proteins (HPF1 for PARP1 and Cullin-4A/RBX for UV-DDB). AIM 2: To define DNA damage processing and handoff at oxidative lesions including single strand breaks and 8-oxoG. Subaim 2a will study the interactions of PARP1, XPC-RAD23B, and UV-DDB on 8-oxoG and single-strand breaks at the single-molecule level. Each protein will be examined individually and in pairs for each damaged substrate. Subaim 2b will measure the binding dynamics for each protein for nucleosomes with 8-oxoG and single-strand breaks positioned at three different sites. Subaim 2c will study 8-oxoG introduced specifically into the chromatinized DNA of living cells utilizing a novel chemoptogenetic system (18, 20, 27). Chromatin remodelers associated with decompaction at damage sites will be knocked down and the repair kinetics will be studied for damage sensor proteins, 8-oxoguanine glycosylase, and other downstream repair proteins.