O-GlcNAcylation of High Mobility Group Box 1 (HMGB1) Alters Its DNA Binding and DNA Damage Processing Activities

Abstract

Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein’s endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein–DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.

Methods for Studying Site-Specific O-GlcNAc Modifications: Successes, Limitations, and Important Future Goals

“… 79 We also used these techniques to study the effect of O-GlcNAcylation on semisynthetic HMGB1 modified at positions Ser100 and demonstrated the influence O-GlcNAc has on HMGB1-DNA interactions. 80 We showed that the PTM generally enhanced the interactions between the protein and DNA and resulted in error-prone repair of ICL-damaged plasmids in U2OS cell extracts. Finally, we have also studied the effect of O-GlcNAc modifications of small heat shock proteins (sHSPs) using NCL and EPL methods.…”

“…We further showed that the PTM is protective against the protein’s cleavage by calpain, and that the anti-aggregation phenotypes imparted by the GlcNAc moiety are unique and not reproduced by other sugars . We also used these techniques to study the effect of O-GlcNAcylation on semisynthetic HMGB1 modified at positions Ser100 and demonstrated the influence O-GlcNAc has on HMGB1-DNA interactions . We showed that the PTM generally enhanced the interactions between the protein and DNA and resulted in error-prone repair of ICL-damaged plasmids in U2OS cell extracts.…”

Exploration of O-GlcNAc-transferase (OGT) glycosylation sites reveals a target sequence compositional bias

“…Amino acids conducive to maintaining an extended or random coil structure significantly affect the propensity of a peptide to be glycosylated and may explain the preference for prolines and beta-branched residues(2). Although the vast majority of OGT substrates are in IDRs(5), there are a few examples of proteins that are glycosylated in folded or ordered regions, including HBGB-1(74), H2B(75) and αB-Crystallin(76). Glycosylation of these ordered sites could occur if the TPR is able to move away from the catalytic site, as has been suggested by a recent electron microscopy structure(77).…”

DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing N6-methyladenosine binding

“…During DNA synthesis, flap endonuclease 1 (FEN1) is O-GlcNAcylated and the abrogation of which will lead to DNA damage accumulation (12). Furthermore, High mobility group B1 (HMGB1) is O-GlcNAcylated, which alters its DNA binding ability and promotes an error-prone DNA repair (13). Recently, WD repeat and HMG-box DNA-binding protein 1 (WDHD1/AND-1) is also shown to be O-GlcNAcylated and takes part in HR (14).…”