DNA-dependent protein kinase catalytic subunit DNA-PKcs/PRKDC is the largest serine/threonine protein kinase of the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family and is the most highly expressed PIKK in human cells. With its DNA-binding partner Ku70/80, DNA-PKcs is required for regulated and efficient repair of ionizing radiation-induced DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. Loss of DNA-PKcs or other NHEJ factors leads to radiation sensitivity and unrepaired DNA double-strand breaks (DSBs), as well as defects in V(D)J recombination and immune defects. In this review, we highlight the contributions of the late Dr. Carl W. Anderson to the discovery and early characterization of DNA-PK. We furthermore build upon his foundational work to provide recent insights into the structure of NHEJ synaptic complexes, an evolutionarily conserved and functionally important YRPD motif, and the role of DNA-PKcs and its phosphorylation in NHEJ. The combined results identify DNA-PKcs as a master regulator that is activated by its detection of two double-strand DNA ends for a cascade of phosphorylation events that provide specificity and efficiency in assembling the synaptic complex for NHEJ.

DNA damage can be introduced by intrinsic or extrinsic stimuli and threaten the transmission of genetic information and ultimately, survival. Eukaryotes have evolved a sophisticated mechanism, DNA damage response (DDR), to counteract this threat posed by DNA damage. The DDR requires a series of proteins involved in multiple DNA repair pathways, of which ATM, ATR and DNA–PK serve as the three most critical kinases. The signaling mechanisms underlying these pathways have been extensively studied and these three master kinases structures determined by cryo-electron microscopy (cryo-EM) have led to tremendous progress in understanding the molecular mechanism of the DDR. In this review, we discuss the recent advances made in understanding the structures of ATM, ATR, and DNA–PK. We highlight the implications of these structures in terms of their function and regulation, and speculate on the critical role of PIKK regulatory domain (PRD) in their activation.

Microproteins, less than 200 amino acids, have been usually regarded as non-functional for a long time. We do not recognize this “dark matter” in the proteome until developing new bioinformatic, biochemical and proteomic techniques. The fast-growing number of the identified microproteins and their emerging roles in various biological processes begin to attract more and more attention. Here, we summarize the recent progress in this field mainly from four aspects: (1) brief history and various definitions of microproteins; (2) three main strategies to identify microproteins; (3) multiple regulatory mechanisms of microproteins; (4) biological functions of microproteins, with a particular focus on genome stability maintenance and their relevance to human diseases. Finally, we discuss some open questions and therapeutic opportunities hiding in these small newcomers.

NAD dependent histone deacetylase SIRT1 has demonstrated involvement in the regulation of stress responses, cellular metabolism, and cell survival. SIRT1 overexpression has been demonstrated to induce G1 arrest, but its function in the cell cycle remains unclear. Here, we identified RecQL4 as a SIRT1 interacting protein through complex purification. RecQL4 is a member of the RecQ DNA helicase family involved in DNA replication, recombination, and repair. Mutations in the RECQL4 gene are responsible for Rothmund–Thomson syndrome (RTS), a severe autosomal recessive disorder causing premature aging and predisposition to cancers. RecQL4 can be acetylated by CBP at lysine 88. Transfection of wild-type RecQL4 into cells derived from an RTS patient can rescue cell proliferation, while a RecQL4 acetylation mutant severely impairs this function. We demonstrated that the acetylation of RecQL4 can regulate both DNA replication activity and the timing of replication firing by dynamically regulating its nuclear localization during the S phase. SIRT1 deacetylates RecQL4 both in vitro and vivo. The acetylation status of RecQL4 affects its loading to the chromatin during the S phase of the cell cycle, consequently affecting DNA replication initiation. Our findings provided new insights on the role of protein acetylation in regulating DNA replication initiation.

N-acetyltransferase 10 catalyzes RNA N4-acetylcytidine (ac4C) modifications and thus regulates RNA stability and translation efficiency. However, the deacetylase for ac4C is unknown. SIRT7 was initially identified as an NAD⁺-dependent protein deacetylase and plays essential roles in genome stability, circadian rhythms, metabolism, and aging. In this study, we identified SIRT7 as a deacetylase of the ac4C of ribosomal (r)RNA for the first time and found it to be NAD⁺-independent. Our data highlight the important role of SIRT7 in rRNA ac4C modification and suggest an additional epitranscriptional regulation of aging.

A correction to this paper has been published: https://doi.org/10.1007/s42764-021-00037-y