Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells

Abstract

Accurate DNA replication during S-phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication-impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non-B DNA, for example, G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y-family (pol eta, pol iota, and pol kappa), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol eta or Pol kappa, but not Pol iota, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harboring multiple copies of a G-rich sequence from the promoter region of the human c-MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol kappa only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL-2 gene and the GA rich region from the far right-hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol eta and pol kappa. We discuss these data in the light of their downregulation in human cancers.

Figure 1. Validation of the RNA interference-based knockdown cellular models (A–C)

We generated U2OS clones expressing shRNA control sequences (shC1 and shC2) and targeting BACH1 (BRIP1) (shB1 and shB2), POL I (shι1 and shι2) or POL H (shη1 and shη2). Extracts from these cells were analyzed by immunoblotting with the indicated antibodies and Ku70 as a loading control (D) U2OS cells were transfected with control siRNA (Luciferase) or two independent Pol κ siRNAs (siκ1 and siκ2). The levels of Pol κ was analyzed by Western blotting 48h after transfection and normalized against actin.

Figure 2. Pol η and Pol κ depletion sensitizes cells to TMS

Cell survival after one week treatment with a range of TMS concentrations for U2OS cell lines depleted for BACH1, Pol η, Pol ι, or Pol κ. All data were performed in triplicate and were taken from the mean survival values obtained from three independent experiments (error bars = standard deviation).

Figure 3. Hela cells transfected with B-DNA and non-B DNA used in this study

Whole cell extracts from Hela cells containing c-MYC-DNA transiently transfected with siRNA against Luciferase (siC), Pol η (siη1 and siη2) or Pol κ (siκ1 and siκ2) were probed by Western blotting with antibodies against Pol η, Pol κ, or Ku70 (as a loading control).

Figure 4. Pol H and Pol K gene silencing triggers H2AX phosphorylation in HeLa-MYC cells

HeLa cells containing B-DNA or MYC-DNA were transfected with control siRNA (Luciferase), two independent Pol κ siRNAs (siκ1 and siκ2) or two independent Pol η siRNAs (siη1 and siη2). Quantification of γ-H2AX-positive cells in the population of each cell line was performed by Flow Cytometry Analysis as described in Materials and Methods. An example is given in (A) where a scatter plot is presented with γ-H2AX intensity on the y axis and propidium iodide intensity on the x axis for the control B-DNA cells and the MYC-DNA cells depleted for Pol κ. The cell populations used for the measurement of the γ-H2AX-positive cells were enclosed by rectangles. (B) Quantification of the FACS analysis were performed as shown in (A) with the cell lines transfected with the indicated siRNAs. The error bars indicate standard deviations of three independent experiments

Figure 5. Depletion of Pol κ, but not that of Pol η, increases H2AX phosphorylation in HeLa-Mbr and HeLa-GA cells

HeLa cells containing B-DNA, Mbr-DNA or GA-DNA were transfected with control siRNA (Luciferase), two independent Pol κ siRNAs (siκ1 and siκ2) or two independent Pol η siRNAs (siη1 and siη2). Quantification of γ-H2AX-positive cells was achieved as in Fig 4. The error bars indicate standard deviations of three independent experiments

Figure 6. mRNA expression of Pol η in the 74 colorectal-adenocarcinomas

Expressions of Pol η as well as the replicative DNA polymerase Pol ε have been measured in the matched human tumoral and normal samples. T (tumoral)/N (normal) mRNA expression ratios between normalized values were calculated. T/N values lower than 1 were transformed to the inverse value N/T. + and − indicate a higher or lower expression respectively in the tumour versus adjacent control tissue. Black boxes above the x axis point out the sample numbers. P-values from bilateral exact binomial test are given uncorrected, the significance level is evaluated using the Benjamini Krieger Yekutieli 2001 procedure for and overall FDR of 0.05. All computations were performed using Stata 9.0 SE (Tiermips).