Biology subjects

Kelch, B. A.

Publications and source records attributed to Kelch, B. A..

3 recordsLinked to original sources

bioRxiv2017

A hyperthermophilic phage decoration protein suggests common evolutionary origin with Herpesvirus Triplex proteins and an anti-CRISPR protein

Virus capsid proteins reproducibly self-assemble into regularly-shaped, stable shells that protect the viral genome from external environmental assaults, while maintaining the high internal pressure of the tightly packaged viral genome. To elucidate how capsids maintain stability under harsh conditions, we investigated the capsid components of a hyperthermophilic virus, phage P74-26. We determined the structure of a capsid protein gp87 and show that it has the same fold as trimeric decoration proteins that enhance the structural stability of capsids in many other phage, despite lacking significant sequence homology. We also find that gp87 is significantly more stable than its mesophilic homologs, reflecting the high temperature environment in which phage P74-26 thrives. Our analysis of the gp87 structure reveals that the core domain of the decoration protein is conserved in trimeric capsid components across numerous dsDNA viruses, including human pathogens such as Herpesviruses. Moreover, this core {beta}-barrel domain is found in the anti-CRISPR protein AcrIIC1, which suggests a mechanism for the evolution of this broad spectrum Cas9 inhibitor. Our work illustrates the principles for increased stability of a thermophilic decoration protein, and extends the evolutionary reach of the core trimeric decoration protein fold.

biochemistry↗

bioRxiv2017

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, substrate secondary structure, and pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), and found that A3A binds RNA in a sequence specific manner. Furthermore, A3A bound tighter to its substrate binding motif when in a loop compared to linear oligonucleotide. Our results suggest that the A3A affinity and preference for substrate is modulated by the structure of DNA, and not just its chemical identity. Analysis of previously published co-crystal structures of A3A bound to ssDNA in light of the above findings directed the proposal of a new model for the molecular mechanism underlying A3A sequence preference. On a broader scale, the results of this work not only provide key insights into the mechanism of A3s beneficial roles in the cell, especially in viral restriction, but also into A3s deleterious activity such as in the development of cancer.

biochemistry↗

bioRxiv2016

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

biochemistry↗