Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor.

Dimeric structure of p300/CBP associated factor.

BACKGROUND: p300/CBP associating factor (PCAF, also known as KAT2B for lysine acetyltransferase 2B) is a catalytic subunit of megadalton metazoan complex ATAC (Ada-Two-A containing complex) for acetylation of histones. However, relatively little is known about the regulation of the enzymatic activity of PCAF. RESULTS: Here we present two dimeric structures of the PCAF acetyltransferase (HAT) domain. These dimerizations are mediated by either four-helical hydrophobic interactions or a ß-sheet extension. Our chemical cross-linking experiments in combined with site-directed mutagenesis demonstrated that the PCAF HAT domain mainly forms a dimer in solution through one of the observed interfaces. The results of maltose binding protein (MBP)-pulldown, co-immunoprecipitation and multiangle static light scattering experiments further indicated that PCAF dimeric state is detectable and may possibly exist in vivo. CONCLUSIONS: Taken together, our structural and biochemical studies indicate that PCAF appears to be a dimer in its functional ATAC complex.

Quikgene: a gene synthesis method integrated with ligation-free cloning.

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.