Cazzaniga, C. | bioRxiv Science

bioRxiv2023

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks

In eukaryotes, CDC7 kinase is crucial for DNA replication initiation and has been involved in fork processing and replication stress response. Human CDC7 requires the binding of either one of two regulatory subunits, DBF4 and DRF1, for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated an isogenic set of cell lines deficient in either one of the two CDC7-activating subunits: these cells are viable but present signs of genomic instability, indicating that both DBF4 and DRF1 can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, including partial deficiency in MCM helicase phosphorylation and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, likely mediating most of its functions in unperturbed DNA replication and during replication fork processing upon replication interference.

biochemistry↗

bioRxiv2023

Discovery and characterization of non-canonical E2 conjugating enzymes

E2 conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation is fundamental for maintaining cellular homeostasis and impacts almost all cellular process. By interacting with multiple E3 ligases, E2s direct the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a post-translational modification that specifically targets lysine side chains (canonical ubiquitylation). We used MALDI-TOF Mass Spectrometry to discover and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We employed protein modelling and prediction tools to identify the catalytic determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results join a stream of recent literature that challenges the definition of ubiquitylation as an exquisitely lysine-specific modification and provide crucial insights into the missing E2 element responsible for non-canonical ubiquitylation. TeaserE2 conjugating enzymes (E2s) play a fundamental role in the attachment of ubiquitin to its substrate. Most E2s can form an isopeptide bond between the ubiquitin C- terminus and a lysine present on the substrate. We identified a family of E2s, UBE2Q1 and UBE2Q2, able to target amino acids other than lysine. Currently nothing is known about their mechanism of action and what substrates they are targeting, even though genetic ablation of UBE2Q1 produce substantial infertility in mice. Here we answer the question about what the key residues beneath their peculiar activity are. We discovered that UBE2Q1 target the lysine-free cytoplasmic domain of the Golgi resident protein Beta-1,4-galactosyltransferase 1, providing an interesting precedent for the role of non-canonical ubiquitylation in eukaryotic cells.

biochemistry↗