Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response

Abstract

Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

RanBP2-mediated SUMOylation promotes human DNA polymerase lambda nuclear localization and DNA repair

“…A defect in the proper and functional folding of fusion proteins cannot be ruled out either. Regardless, our study uncovers the pivotal role of SUMOylation in the control of cellular distribution of Polλ, and identify the molecular basis of previous observations that Polλ catalytic core, a protein version lacking the amino terminal region including BRTC and Ser-Pro rich domains, is not able to enter into the nucleus [33,34].…”

Variations in nuclear localization strategies among pol X family enzymes

“…In selecting these test peptides, we made an effort to ensure that the length was sufficiently inclusive to cover residues that might be part of the functional NLS but were not identified by the computational screen. In the case of pol λ, which had only very low scoring NLS sequences, NLS localization to the N‐terminal region of the enzyme is also supported by reported biochemical and localization studies …”

“…A pol μ bipartite NLS sequence identified by NLS Mapper does not bind Impα, and if bound, the overlap of the C‐terminal residues of the identified sequence with the N‐terminus of the pol μ BRCT domain would be structurally problematic, but is in agreement with the initially proposed sequence . The pol λ bipartite NLS complex identified crystallographically was either not identified by some software programs or, as indicated in Table , corresponded to a very low scoring sequence, but was in the N‐terminal region previously shown to contain an NLS . Similarly, we recently determined that pol β, generally thought to lack a NLS, contains a functional NLS at its N‐terminus, and that XRCC1 contains a bipartite NLS separated by an atypically long internal linker that is not readily identified by many NLS software analysis programs .…”

“…Of the four mammalian enzymes, only the pol λ NLS utilizes a bipartite structure and in the cell types investigated, shows the greatest extent of nuclear localization; these results are consistent with earlier reports indicating that the NLS is located in the N‐terminal region of the enzyme . In the pol λ‐Impα complex, the AKIPR motif found in the major binding pocket lacks a basic residue at position P3 that is commonly encountered at this position (Table S2).…”

“…As a consequence of the issues outlined above, we have extended our studies to include the three other mammalian pol X enzymes: DNA polymerase mu (pol μ), DNA polymerase lambda (pol λ), and terminal deoxynucleotidyl transferase (TdT), in order to better understand variations in nuclear import among this family of proteins. Previous reports identifying potential NLS sequences in these proteins were used, as well as identifications based on the software NLS Mapper . NLS sequences and their interactions with ImpαΔIBB (Impα lacking the importin β‐binding domain) were subsequently characterized by X‐ray crystallography and fluorescence polarization binding assays.…”