Dr1 (NC2) is present at tRNA genes and represses their transcription in human cells

Dr1 (also known as NC2{beta}) was identified as a repressor of RNA polymerase (pol) II transcription. It was subsequently shown to inhibit pol III transcription when expressed at high levels in vitro or in yeast cells. However, endogenous Dr1 was not detected at pol III-transcribed genes in growing yeast. In contrast, we demonstrate that endogenous Dr1 is present at pol III templates in human cells, as is its dimerization partner DRAP1 (also called NC2{alpha}). Expression of tRNA by pol III is selectively enhanced by RNAi-mediated depletion of endogenous human Dr1, but we found no evidence that DRAP1 influences pol III output in vivo. A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1. This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction. On the basis of these data, we conclude that the physiological functions of human Dr1 include regulation of pol III transcription

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin

Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function