UC Riverside

The genetic code contains all the information needed to execute every biological task in cells. Although this information is encoded in DNA, proteins are the primary players that execute the biological functions. Understanding how DNA is recognized and regulated by cellular proteins is crucial to gain a deeper understanding of cell biology. Recent advances in mass spectrometry instrumentation allow the investigation of large portions of the proteome simultaneously, making it an attractive technique to examine DNA-Protein interactions. In this thesis, we aimed to develop a quantitative proteomic approach to identify novel nucleic acid-binding proteins.

In chapters 2 and 3, we developed and applied a quantitative proteomic method to identify proteins that recognize non-canonical G-quadruplex (G4) DNA. G4s are important biological players due to their genomic locations and their roles in functional biology. We investigated the interaction proteomes of three unique G4 structures. Our technique facilitated the identification of 84 proteins that preferentially bind to G4, including NSUN2 and SLIRP. Upon further investigation, we demonstrate that NSUN2 is a selective binder of G4 structures derived from the promoter of cMYC and cKIT genes, but not that from the human telomere.

In chapter 3, we characterized the binding of SLIRP to G4 DNA. We demonstrated that SLIRP can selectively bind all three G4 DNA patterns with low nanomolar binding affinity; the protein, however, exhibited significantly lower binding affinity to single-stranded DNA. Using site-directed mutagenesis, we found that L62 and R24/R25 in the RNA recognition motif of SLIRP are key residues for G4 recognition and binding.

In chapter 4, we extended our technique to identify the proteins that interact with two tandem DNA lesions, cdA and cdG. We uncovered 33 proteins can bind to duplex DNA harboring a site-specifically incorporated cdA and/or cdG lesions over their unmodified counterparts. We investigated further one of the putative cdA- and cdG-binding proteins, CDKN2AIP. We knocked out the CDKN2AIP gene and discovered that CDKN2AIP-/- cells displayed significantly poorer survival than wild-type cells when challenged with DNA damaging agents that can induce cdA and cdG lesions, but not those agents that induce other types of DNA lesions.