Different types of interaction between PCNA and PIP boxes

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  • 78987910 Nucleic Acids Research, 2015, Vol. 43, No. 16 Published online 13 July 2015doi: 10.1093/nar/gkv712

    Different types of interaction between PCNA and PIPboxes contribute to distinct cellular functions ofY-family DNA polymerasesYuji Masuda1,2,, Rie Kanao1,, Kentaro Kaji1, Haruo Ohmori3,4, Fumio Hanaoka4 andChikahide Masutani1,*

    1Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho,Chikusa-ku, Nagoya 464-8601, Japan, 2Department of Toxicogenomics, Nagoya University Graduate School ofMedicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan, 3Department of Gene Information, Institute forVirus Research, Kyoto University, Sakyo-ku, Kyoto 606-8517, Japan and 4Department of Life Science, GraduateSchool of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan

    Received April 24, 2015; Revised June 26, 2015; Accepted July 01, 2015


    Translesion DNA synthesis (TLS) by the Y-familyDNA polymerases Pol, Pol and Pol, mediatedvia interaction with proliferating cell nuclear anti-gen (PCNA), is a crucial pathway that protects hu-man cells against DNA damage. We report that Polhas three PCNA-interacting protein (PIP) boxes (PIP1,2, 3) that contribute differentially to two distinctfunctions, stimulation of DNA synthesis and promo-tion of PCNA ubiquitination. The latter function isstrongly associated with formation of nuclear Polfoci, which co-localize with PCNA. We also showthat Pol has two functionally distinct PIP boxes, likePol, whereas Pol has a single PIP box involved instimulation of DNA synthesis. All three polymeraseswere additionally stimulated by mono-ubiquitinatedPCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Pol exert redundant and ad-ditive effects in vivo via distinct molecular mecha-nisms. These findings provide an integrated pictureof the orchestration of TLS polymerases.


    Translesion DNA synthesis (TLS), a DNA damage toler-ance mechanism, is a crucial biological function that pro-tects cells from various genotoxic agents. Particularly in hu-mans, DNA polymerase (Pol), a Y-family DNA poly-merases (1), plays an important role in preventing cell deathand mutagenesis after ultraviolet (UV) light irradiation, andmalfunction of Pol causes the inherited genetic disorder,xeroderma pigmentosum variant (XP-V) (24).

    Interactions between proliferating cell nuclear antigen(PCNA) and the three Y-family human DNA polymerases(Pol, Pol and Pol) are critically involved in regulationof TLS. Pol and Pol are known to contain two PCNA-interacting protein (PIP) boxes (PIP1 and PIP2) in theircentral and C-terminal regions, respectively, whereas Polis known to contain only one functional PIP box (PIP1, inthe central region) (512). In DNA-damaged cells, PCNA ismono-ubiquitinated at residue K164 by the RAD6RAD18complex (1315), and poly-ubiquitinated by additional fac-tors including UBC13, MMS2, and RAD5/HLTF or SH-PRH (13,1619). Each of the three Y-family DNA poly-merases described above has one or two copies of theubiquitin-binding domain (UBD), called UBZ (ubiquitin-binding zinc-finger) in Pol and Pol and UBM (ubiquitin-binding motif) in Pol (6). These findings support the no-tion that mono-ubiquitination of PCNA plays a key role inswitching from replicative DNA polymerase stalled at a siteof DNA damage to a DNA polymerase (such as Pol, or )capable of carrying out TLS (1415,2021). However, thisidea is still controversial (5), and more recent publicationsreport that Pol and Pol are able to carry out TLS inde-pendently of PCNA ubiquitination in some circumstances(2224).

    The intracellular functions of the various motifs of Polare monitored in two ways: formation of nuclear foci con-taining Pol co-localized with PCNA and complementa-tion of UV sensitivity of XP-V cells. Mutations in PIP2strongly impair the localization of Pol, indicating thatPIP2 plays a crucial role in the accumulation of this pro-tein in replication foci (23,2527). However, ubz mutantsalso failed to accumulate in replication foci (6,26), and accu-mulation is barely detectable in a human cell line expressingthe PCNAK164R mutant instead of endogenous PCNA (28)

    *To whom correspondence should be addressed. Tel: +81 52 789 3871; Fax: +81 52 789 3890; Email: masutani@riem.nagoya-u.ac.jpThese authors contributed equally to the paper as first authors.

    C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

    Downloaded from https://academic.oup.com/nar/article-abstract/43/16/7898/1075771by gueston 20 March 2018

  • Nucleic Acids Research, 2015, Vol. 43, No. 16 7899

    and in a PCNAK164R knock-in murine cell line (23), demon-strating that the PIP2PCNA interaction itself is not suffi-cient for foci formation. Recently, Durando and colleaguesreported an additional function of PIP2 of Pol, namely,that Pol promotes mono-ubiquitination of PCNA in aPIP2-dependent manner (29). It remains unclear how thetwo PIP2-mediated activities, co-localization of Pol withPCNA and promotion of PCNA mono-ubiquitination, arelinked at the molecular level.

    The pip2 and ubz mutants of Pol exhibit pronounceddefects in foci formation, but retain the capacity to com-plement UV sensitivity of XP-V cells; however, the levels ofcomplementation activity vary amongst studies by differentgroups (56,2526,30), including one report that showed norole for PIP2 in survival after UV irradiation (27). Even apip2 ubz double mutant (25,26) and a mutant in which PIP2and UBZ were deleted (30) still exhibited significant levelsof complementation activity. By contrast, the pip1 pip2 dou-ble mutant and a mutant lacking the entire PIP1, UBZ andPIP2 domains were severely defective in complementation(5,30).

    The biochemical activities of the motifs of human Polhave been studied in vitro. In primer extension assays, bothPIP1 and PIP2 contributed to some extent to the stimula-tion of DNA synthesis in the presence of PCNA (5,8,30).Using a reconstitution system to investigate switching be-tween Pol and Pol at DNA lesions, we demonstratedthat the weak enhancement of the recruitment of Pol tothe 3-end of primer DNA by the UBZ domain dependedon mono-ubiquitination of PCNA (21). These biochemicalproperties of mutant proteins defective for PIP1, PIP2 orUBZ are well correlated with their abilities to complementUV sensitivity of XP-V cells, but not with their abilities topromote accumulation into the replication foci.

    Here, we describe the molecular functions of Pols PIP1,PIP2 and UBZ domains, together with the newly foundPIP3, in UV tolerance, and present findings that resolve thecontroversies raised in previous reports. Our key finding isthat Polhas two functionally distinct types of PIP box, onethat stimulates DNA synthesis, and another that promotesPCNA mono-ubiquitination and accumulation into repli-cation foci. Both PIP functions, together with the UBZ do-main, are redundantly required for survival after UV irra-diation. Additionally, we show that Pol has two PIP boxeswith different functions, whereas Pol has only one PIP boxinvolved in stimulation of DNA synthesis. Taking the re-sults of previous reports together with the in vivo and invitro data obtained in this study, we propose a model forthe cellular functions of the various motifs of Pol// inorchestrating TLS.



    Expression plasmids were constructed as follows. HumanPOLH, POLI (encoding 740 amino-acid residues) (11) andPOLK were cloned into pET20b(+) (Novagen) to obtainuntagged proteins, and into pET15b (Novagen) to obtainN-terminally histidine-tagged proteins. Plasmids for expres-sion of Pol, Polubz and His-Pol in E. coli were described

    previously (21,31). A truncated gene encoding PolC (32)was cloned into pET21a(+) (Novagen). A gene encodingUBCH5cS22R was cloned into pET15b. Mutations were cre-ated by PCR, and nucleotide sequences were verified aftercloning.

    E1, RAD6-(His-RAD18)2, RAD6-(RAD18C2)2, ubiq-uitin, RPA, PCNA, RFC, His-Pol, Pol and their mutantswere purified as described previously (21,31,3335). Col-umn chromatography was carried out at 4C on an FPLCsystem (GE Healthcare Life Science) using columns fromGE Healthcare unless otherwise indicated. Protein concen-trations were determined by the Bio-Rad protein assay us-ing BSA (Bio-Rad) as the standard.

    PolC was purified in the same way as Pol (21), exceptthat HiTrap Phenyl HP was used instead of an Econopackmethyl column (Bio-Rad).

    A histidine-tagged Pol (His-Pol) and its mutants werepurified as follows. BL21 (DE3) harbouring each of the ex-pression plasmids and pMS-tRNA1 (36) was grown in 2l of Terrific broth (37) supplemented with ampicillin (250g/ml) and kanamycin (30 g/ml) at 15C. His-Pol wasinduced with 0.2 mM isopropyl -D-thiogalactopyranoside(IPTG) for 8 h, and then purified by sequential chromatog-raphy on Ni2+-charged HiTrap chelating HP, POROS 50HE (Applied Biosystems), Econopack CHT-II (BIO-RAD)and Superdex 200 columns. The peak fraction containingHis-Pol was frozen in liquid nitrogen and stored at 80C.

    Pol and Polpip1 were purified as follows. BL21 (DE3)harbouring each of the expression plasmids was grown in 5l of LB supplemented with ampicillin (250 g/ml) at 15C.Pol was induced with 0.2 mM IPTG for 5 h, and then puri-fied by sequential chromatography on HiTrap Capto MMC,Ni2+-charged HiTrap chelating HP, HiTrap SP HP, HiTrapQ HP and Superdex 200 columns. Note that Pol itself (with-out the His-tag) has weak affinity for the Ni2+-charged Hi-Trap chelating column. For Polpip1, the gel-filtra