Deubiquitinating enzymes and the proteasome regulate preferential sets of ubiquitin substrates.

The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Here, we compare the contributions of the proteasome and DUBs on the global ubiquitinome, using UbiSite technology, inhibitors and mass spectrometry. We uncover large dynamic ubiquitin signalling networks with substrates and sites preferentially regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitination.

Methods to Study Intracellular Movement and Localization of the Nucleotide Excision Repair Proteins at the DNA Lesions in Mammalian Cells.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. The mammalian NER utilizes more than 30 proteins, in a multi-step process that begins with the lesion recognition within seconds of DNA damage to completion of repair after few hours to several days. The core proteins and their biochemical reactions are known from in vitro DNA repair assays using purified proteins, but challenge was to understand the dynamics of their rapid recruitment and departure from the lesion site and their coordination with other proteins and post-translational modifications to execute the sequential steps of repair.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site.

Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro.

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation.

Resistance to PARP-Inhibitors in Cancer Therapy.

The pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) family of proteins have shown promising results in preclinical studies and clinical trials as a monotherapy or in combination therapy for some cancers. Thus, usage of PARP-inhibitors (PARPi) in cancer therapy is bound to increase with time, but resistance of cancer cells to PARPi is also beginning to be observed. Here we review different known and potential mechanisms by which: (i) PARPi kill cancer cells; and (ii) cancer cells develop resistance to PARPi.

Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair.

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV.