DNA-Protein Cross-links: Formation, Repair, and Inhibition of DNA replication

Abstract

DNA-protein crosslinks (DPCs) are ubiquitous DNA lesions that form when cellular proteins become trapped on DNA following exposure to UV light, free radicals, aldehydes, and transition metals. These ultra-bulky lesions are known to disrupt regular DNA cellular machinery, such as replication, transcription, and repair, leading to mutagenesis and carcinogenesis. DPCs can also form endogenously when naturally occurring epigenetic marks (5-formyl cytosine, 5fC) in DNA react with lysine and arginine residues of histones H2A, H3, and H4 to form Schiff base conjugates. However, the understanding of cellular effects on DPCs is not fully understood. The main objective of this thesis was to investigate the effects of DPCs on replication, as well as elucidate mechanisms of repair.In Chapter II, we investigated the local DNA sequence effects on TLS polymerase bypass of 5fC-mediated DNA-peptide crosslinks. Our previous studies revealed that full size DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that when placed in 5'-CXA-3' sequence context (X=5fC-peptide lesion), DNA-peptide crosslinks can be bypassed by human translesion synthesis (TLS) polymerases ƞ and k in an error-prone manner. However, local nucleotide sequence context can have a large effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex between DNA template, polymerase, and the incoming dNTP. In this chapter, model hydrolytically stable DpCs were prepared by oxime ligation between 5fC in DNA and oxy-lysine containing peptides. Primer extension products were analyzed by gel electrophoresis, and steady state kinetics of dAMP incorporation opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase ƞ activity both in the absence and presence of a DpC lesion. Molecular modeling and molecular dynamics simulations of the hPol η ternary complex, containing the DNA template-primer strands with incoming dATP opposite DpC or unmodified C explained structurally how the nature of the 5' and 3' neighbors of this template profoundly impacted its alignment in the C-A mismatch. Our results reveal an important role of base sequence context in promoting TLS related mutational hotspots both in the presence and in the absence of DpC lesions. In Chapter III, we investigated the role of replicative DNA polymerases δ and ε in DpC lesion bypass. TLS polymerase switches are known to be the primary mechanism to bypass bulky DNA lesions such as DNA-peptide crosslinks, however, DpC-containing plasmids were still replicated at relatively high efficiency in TLS-deficient cell lines, leading to the hypothesis that replicative polymerases are also involved in lesion bypass, in a minor role. In Chapter IV, we employed a sensitive nanoLC-ESI+-LC-MS/MS assay to investigate the formation of ROS-induced DPCs between thymidine in DNA and tyrosine in proteins. This methodology was used to analyze the role of metalloprotease Spartan in repair of ROS-induced DPCs in cells and mouse tissues. A 1.5-2 fold increase of thymidine-tyrosine adducts were detected in the brain, heart, livers, and kidneys of Spartan hypomorphic (SPRTNf/-) mice compared to wild type (SPRTN+/+), providing evidence that Spartan plays a direct role in the repair of ROS-induced DPCs.