Expression and Structural Analyses of Human DNA Polymerase ? (POLQ).

ABSTRACT: DNA polymerase theta (pol ?) is an evolutionarily conserved protein encoded by the POLQ gene in mammalian genomes. Pol ? is the defining enzyme for a pathway of DSB repair termed "alternative end-joining" (altEJ) or "theta-mediated end-joining." This pathway contributes significantly to the radiation resistance of mammalian cells. It also modulates accuracy in repair of breaks that occur at stalled DNA replication forks, during diversification steps of the mammalian immune system, during repair of CRISPR-Cas9, and in many DNA integration events. Pol ? is a potentially important clinical target, particularly for cancers deficient in other break repair strategies. The enzyme is uniquely able to mediate joining of single-stranded 3' ends. Because of these unusual biochemical properties and its therapeutic importance, it is essential to study structures of pol ? bound to DNA. However, challenges for expression and purification are presented by the large size of pol ? (2590 residues in humans) and unusual juxtaposition of domains (a helicase-like domain and distinct DNA polymerase, separated by a region predicted to be largely disordered). Here we summarize work on the expression and purification of the full-length protein, and then focus on the design, expression, and purification of an active C-terminal polymerase fragment. The generation of this active construct was nontrivial and time consuming. Almost all published biochemical work to date has been performed with this domain fragment. Strategies to obtain and improve crystals of a ternary pol ? complex (enzyme:DNA:nucleotide) are also presented, along with key elements of the structure.

SUBMITTER: Malaby AW 

PROVIDER: S-EPMC5624038 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature