The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery

Abstract Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.

EST003 using oligonucleotides oEST041 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST115: Pol Y1 loxP spec.The yqjH gene and upstream were amplified from strain EST003 using oligonucleotides oEST023 and oEST070.The loxP spec cassette was amplified from strain EST081 using oligonucleotides oEST032 and oEST008.The yqjH downstream was amplified from strain EST003 using oligonucleotides oEST071 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST117: ΔPol Y2.The yqjW upstream was amplified from strain EST003 using oligonucleotides oEST049 and oEST072.The loxP spec cassette was amplified from strain EST081 using oligonucleotides oEST032 and oEST008.The yqjW downstream was amplified from strain EST003 using oligonucleotides oEST073 and oEST048.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST119: Pol Y1-CBM-mut1.The N-terminal portion of yqjH (from the N-terminus to the clampbinding motif) and the upstream were amplified from strain EST115 using oligonucleotides oEST023 and oEST058.The C-terminal portion of yqjH (from the clamp-binding motif to the Cterminus) and the downstream were amplified from strain EST115 using oligonucleotides oEST059 and oEST028.Oligonucleotides oEST058 and oEST059 contained the CBM-mut1 mutations.The two fragments were joined by Gibson assembly and transformed into strain EST003.
EST121: Pol Y1-CBM-mut2.The N-terminal portion of yqjH (from the N-terminus to the clampbinding motif) and the upstream were amplified from strain EST115 using oligonucleotides oEST023 and oEST060.The C-terminal portion of yqjH (from the clamp-binding motif to the Cterminus) and the downstream were amplified from strain EST115 using oligonucleotides oEST061 and oEST028.Oligonucleotides oEST060 and oEST061 contained the CBM-mut2 mutations.The two fragments were joined by Gibson assembly and transformed into strain EST003.
EST139: Pol Y1-Dendra2.The yqjH gene and upstream were amplified from strain EST003 using oligonucleotides oEST023 and oEST029.The linker and Dendra2 were amplified from strain EST081 using oligonucleotides oEST033 and oEST034.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST141: Pol Y1-mYPet.The yqjH gene and upstream were amplified from strain EST003 using oligonucleotides oEST023 and oEST029.The linker and mYPet were amplified from strain EST057 using oligonucleotides oEST035 and oEST036.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST143: Pol Y1-Halo.The yqjH gene and upstream were amplified from strain EST003 using oligonucleotides oEST023 and oEST029.The linker and HaloTag were amplified from strain EST083 using oligonucleotides oEST030 and oEST031.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST191: Pol Y1-cat-mut.The N-terminal portion of yqjH (from the N-terminus to the catalytic site) and the upstream were amplified from strain EST115 using oligonucleotides oEST037 and oEST062.The C-terminal portion of yqjH (from the clamp-binding motif to the C-terminus) and the downstream were amplified from strain EST115 using oligonucleotides oEST063 and oEST028.Oligonucleotides oEST062 and oEST063 contained the catalytic site mutations.The two fragments were joined by Gibson assembly and transformed into strain EST003.
EST215: Pol Y1-CBM-mut1-Halo.The yqjH gene (containing the CBM-mut1 mutation) and upstream were amplified from strain EST119 using oligonucleotides oEST023 and oEST029.The linker and HaloTag were amplified from strain EST083 using oligonucleotides oEST030 and oEST031.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.
EST217: Pol Y1-CBM-mut2-Halo.The yqjH gene (containing the CBM-mut2 mutation) and upstream were amplified from strain EST121 using oligonucleotides oEST023 and oEST029.The linker and HaloTag were amplified from strain EST083 using oligonucleotides oEST030 and oEST031.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.EST229: Pol Y1-cat-mut-Halo.The yqjH gene (containing the catalytic site mutations) and upstream were amplified from strain EST191 using oligonucleotides oEST023 and oEST029.The linker and HaloTag were amplified from strain EST083 using oligonucleotides oEST030 and oEST031.The loxP spec cassette and yqjH downstream were amplified from strain EST115 using oligonucleotides oEST032 and oEST028.The three fragments were joined by Gibson assembly and transformed into strain EST003.

Figure S2 .
Figure S2.Cellular localization of DnaX-mYPet, Pol Y1-Halo, and the nucleoid label HBsu-mYPet.Long cell axis projections of Pol Y1 (left) and false positive spots (right) in (A) untreated cells and (B) cells treated with 10 M 4-NQO for 1 h on a per cell basis.Long and short cell axis projections of DnaX in untreated cells either (C) labeled with 2.5 nM JFX554 or (D) unlabeled.Long cell axis projections of the nucleoid label HBsu and (E) all Pol Y1 trajectories or (F) static Pol Y1 trajectories (D * < 0.14 m 2 /s) in untreated cells.Panel (C) is reproduced from Figure 3B.

Figure S3 .
Figure S3.Cellular localization of DnaX-mYPet and Pol Y1-Halo.Long and short cell axis projections of (A, B) DnaX and (C, D) Pol Y1 in untreated cells and cells treated with 10 M 4-NQO for 1 h, respectively, recorded with a long 250 ms integration time.Long and short cell axis projections of Pol Y1 in cells treated with (E) 1 M 4-NQO for 1 h and (F) 10 M 4-NQO for 2 h.

Figure S6 .
Figure S6.Apparent diffusion coefficient (D * ) distributions for Pol Y1-Halo and corresponding three-population fits.D * distributions for WT Pol Y1 (A, C) and false positive spots (B, D) in untreated cells and cells treated with 10 M 4-NQO for 1 h, respectively, on a per cell basis.D * distributions for WT Pol Y1 in cells treated with (E) 1 M 4-NQO for 1 h and (F) 10 M 4-NQO for 2 h.D* distributions for (G) Pol Y1-CBM-mut1, (H) Pol Y1-CBM-mut2, and (I) Pol Y1-catmut mutants in cells treated with 10 M 4-NQO for 1 h.(J) D * distributions for WT Pol Y1 in untreated cells with a two-population instead of three-population fit.

Figure S8 .
Figure S8.Pol Y1-Halo binding lifetime measurements.(A) Cartoon of Pol Y1 dissociation and JFX554 photobleaching pathways.(B) Apparent Pol Y1 binding lifetime for WT Pol Y1 in untreated cells, Pol Y1-cat-mut in untreated cells, and WT Pol Y1 in cells fixed with formaldehyde.(C) Apparent Pol Y1 binding lifetime for WT Pol Y1 in untreated cells, cells treated with 10 M 4-NQO for 1 h, and cells fixed with formaldehyde.Distributions of apparent binding lifetime and corresponding exponential fits for (D) WT Pol Y1 in fixed cells, (E) WT Pol Y1 in untreated cells, (F) Pol Y1-cat-mut in untreated cells, and (G) WT Pol Y1 in cells treated with 10 M 4-NQO for 1 h.(Note that the y-axes are truncated in D -G to show the longer timescale behavior more clearly.)