Research Article: Impact of the DNA Damage Response on Human Papillomavirus Chromatin

Date Published: June 16, 2016

Publisher: Public Library of Science

Author(s): Dipendra Gautam, Cary A. Moody, Rebecca Ellis Dutch.


Partial Text

The fidelity of replication is regulated by the DNA damage response (DDR), an elaborate signaling network of proteins that detect, signal, and repair DNA lesions. While some viruses have evolved mechanisms to avoid or eliminate DNA repair machinery, others exploit the DDR to replicate their genomes [1]. Recent studies indicate that the DDR facilitates productive replication of human papillomaviruses (HPV) [2–8]. The ability of cells to detect and repair DNA breaks is dependent on the reorganization of surrounding chromatin [9]. The importance of histone post-translational modifications and chromatin remodeling proteins in recruitment of repair factors to DNA breaks is becoming increasingly clear. HPV genomes are histone-associated in the virion and exhibit a nucleosome pattern similar to that of cellular DNA in infected cells [10,11]. HPV chromatin is subject to histone modifications, likely important in ensuring the correct temporal expression of viral genes through the life cycle [12,13]. However, the assembly of DNA repair factors in large complexes at HPV replication centers raises the intriguing possibility that viral chromatin may also be subject to the changing chromatin dynamics associated with the DDR, facilitating efficient productive replication through DNA repair mechanisms.

HPVs are small, double-stranded DNA viruses that exhibit a strict tropism for the mucosal or cutaneous stratified squamous epithelium. Mucosal HPV types are grouped into high-risk and low-risk categories based on their association with cancer. Outcomes of HPV infection can range from asymptomatic to a wide range of benign papillomas or warts. However, high-risk HPV types are the etiological agent of cervical cancer and other anogenital malignancies as well as an increasing number of oropharyngeal cancers [14].

ATM is a serine/threonine kinase belonging to the PIKK family, which also includes DNA-PK (DNA-dependent protein kinase) and ATR (ATM and Rad3-related) [19]. ATM and DNA-PK are activated primarily in response to double-strand breaks (DSBs), while ATR responds to single-stranded DNA (ssDNA) that occurs upon resection of DSBs, or results from stalled replication forks. Once activated, these kinases initiate a signal transduction cascade resulting in activation of cell cycle checkpoints and recruitment of DNA repair factors to damaged DNA [20]. A seminal study in the HPV field demonstrated that ATM activation is required for productive replication of high-risk HPV31, but not for episomal maintenance [2]. Subsequent studies demonstrated that components of the ATM response are recruited to HPV replication sites (H2AX, Chk2, RPA, MRN complex [Mre11, Rad50, Nbs1], 53BP1, BRCA1, Rad51) [4,5,21–23], suggesting that HPV utilizes ATM activity to drive productive replication through DSB repair mechanisms. Both E7 and the viral helicase E1 can independently activate the ATM response and may have distinct roles in maintaining ATM activity in HPV-infected cells during various stages of the viral life cycle [2,21,22,24]. Several studies have shown that the ATR pathway is also active in HR-HPV-positive cells and can be activated in an E7- or E1-dependent manner [2,8,21,24]. ATR and its effector kinase Chk1 are required to stabilize replication forks in response to replication stress. Multiple factors from the ATR pathway localize to HPV replication compartments [21,24,25], and recent studies demonstrated that inhibition of ATR and Chk1 blocks productive replication [8]. Overall, these studies suggest that HPV manipulates both the ATM and ATR arms of the DDR in order to promote viral genome stability and ensure the efficient amplification of viral genomes through DNA repair mechanisms.

DNA damage induces structural changes in chromatin that are orchestrated through ATP-dependent remodeling complexes as well as post-translational modifications of histones and histone-binding proteins (i.e., phosphorylation, acetylation, ubiquitylation) [9,20]. The general chromatin response to DSB formation is outlined in Fig 1 and discussed briefly below. In response to DSBs, ATM is activated via recruitment to DNA lesions by the MRN complex and acetylation by TIP60 [28]. At DSBs, ATM rapidly phosphorylates the histone variant H2AX on S139, forming γH2AX [29]. γH2AX initiates the assembly of repair factors at DNA lesions in a highly regulated manner, with one key function being the recruitment of the scaffolding protein MDC1. MDC1 recruits the MRN complex, further amplifying the DDR response. MDC1 also promotes recruitment of the E3 ubiquitin ligases Ring Finger 8 (RNF8) and RNF168. Together, RNF8/RNF168 catalyze non-proteolytic K63-linked ubiquitin chains on H2A/H2AX, facilitating the binding of BRCA1 as well as 53BP1 [30]. The interplay between 53BP1 and BRCA1 fine-tunes the DSB repair pathway utilized, with BRCA1 promoting HR through initiating end resection in S/G2 and 53BP1 committing repair to NHEJ by blocking BRCA1 accumulation and end resection in G1 [26].

Several recent studies support the idea that HPV chromatin is modified by the DDR. Gillespie et al. demonstrated that γH2AX localizes to HPV replication compartments, with γH2AX foci size increasing with productive replication [4]. Importantly, γH2AX was found to bind viral DNA, suggesting that γH2AX may serve to assemble repair factors at viral replication sites. In support of this, DDR components that rely on γH2AX for recruitment to DNA breaks, including 53BP1, Nbs1, BRCA1, and Rad51, also localize to HPV replication compartments [4,22,23,36]. Given that the recruitment of 53BP1 as well as BRCA1 to DSBs can occur in an ubiquitin-dependent manner, these results also suggest that RNF8/RNF168 may localize to viral DNA. However, the impact of HPV infection on RNF8/RNF168 expression, localization, and function has not been determined.

HPV requires ATM activity and the recruitment of HR factors to viral DNA for productive replication. The binding of γH2AX to viral DNA suggests that HPV-induced activation of ATM results in chromatin changes that promote the recruitment of HR rather than NHEJ factors to viral replication centers. Understanding how viral chromatin modifications are altered by the DDR and whether this deviates from the normal response to DNA damage will provide further insight into the mechanisms by which viral replication is controlled. Activation of ATM, phosphorylation of H2AX, and the recruitment of DNA repair factors to viral replication centers are observed upon infection with multiple DNA viruses, including SV40, HCMV, HSV-1, KSHV, EBV, MCPyV, and γHV68 [40,41]. Determining if DDR-associated changes to viral chromatin serve as a common means to facilitate the recruitment of repair factors to viral DNA and promote viral replication provides an exciting avenue of future investigation.




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