The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation - PubMed (original) (raw)

The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation

H Zan et al. Immunity. 2001 May.

Abstract

Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol zeta, which effectively extends DNA past mismatched bases (mispair extender), and pol eta, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4+ T cells, these lymphocytes upregulated pol zeta, downregulated pol eta, and mutated the Ig and bcl-6 genes. Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol zeta plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol eta.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Somatic Hypermutating Human Tonsillar B Cells Upregulate DNA pol ζ and Downregulate pol η

Mutating IgD+CD38+ early centroblasts (EC) and IgD−CD38+ centroblasts/centrocytes (CC) and unmutating IgD+CD38− naive (N) and IgD−CD38− memory (M) B cells were isolated from human tonsillar B cells and analyzed for the expression of pol ζ REV3, pol η (RAD30A), pol ι (RAD30B), pol α, pol β, pol δ, pol ε, pol κ, and pol μ, using RT-PCR. Pol ζ, pol η, and β-actin were amplified in the same vessel. These findings were derived from one of three experiments yielding comparable results.

Figure 2

Figure 2. Upregulation of pol ζ and Downregulation of pol η Are Induced by BCR Engagement and Are Associated with Somatic Hypermutation

Human IgM+IgD+ CL-01 cells were cultured in the presence or absence of activated CD4+ T cells with or without treatment with anti-BCR Ab. After 12 hr, 1, 2, 5, 7, and 9 days, 0.5 × 106 B cells from each culture were harvested. RNA was extracted and used to analyze the expression level of pol ζ REV3, pol η, pol ι, pol α, pol β, pol δ, pol ε, and pol μ by RT-PCR. Shown are the levels of the DNA polymerases after 12 hr of culture. Comparable levels were detected at day 1, 2, 5, 7, and 9. After 14 days, the cultures were terminated to identify and analyze the Ig VHDJH mutated transcripts. Histograms depict the findings derived from one of three independent experiments yielding comparable results.

Figure 3

Figure 3. Pol ζ Is Upregulated and pol η Is Downregulated by BCR but Not TCR in a Dose- and Time-Dependent Fashion in Human Lymphocytes

(A) Human CL-01 cells were reacted with increasing amounts of anti-BCR Ab for 1 hr at 4°C, washed, and cultured in FCS-RPMI for 12 hr. The level of pol ζ REV3, pol η, pol ι, pol α, pol β, pol δ, pol ε, and pol μ was measured by RT-PCR for each anti-BCR Ab concentration. (B) Level of pol ζ REV3, pol η, and β-actin in the same B cells as in (A), as determined by single-vessel RT-PCR incorporating [α-32P]dCTP and consisting of 10, 15, or 25 cycles. (C) Human CL-01 cells were reacted with anti-BCR Ab, washed, and cultured in FCS-RPMI. The level of pol ζ, pol η, and pol ι was measured at 1, 3, 6, 9, 24, and 48 hr. (D) Freshly isolated normal human peripheral blood IgM+IgD+ B cells were reacted with anti-BCR Ab (1 μg/ml) for 1 hr at 4°C, washed, and cultured in FCS-RPMI for 12 hr before analysis of pol ζ, pol η, and pol ι transcripts. (E) Normal human CD4+ normal T cells were cultured in FCS-RPMI in flat-bottom 96-well culture plates coated with 1:800 OKT3 anti-CD3 Ab for 12 hr and then analyzed for pol ζ, pol η, and pol ι transcripts.

Figure 4

Figure 4. Specific Inhibition of pol ζ REV3 Expression Impairs Ig VHDJH and bcl-6 Somatic Hypermutation as well as Damage-Induced DNA Mutagenesis in CL-01 Cells Induced by BCR Engagement and Contact with Activated CD4+ T Cells

(A) Human B cells cocultured with activated CD4+ T cells upon BCR engagement after daily treatment with Cytofectin GSV only (Nil) or Cytofectin GSV together with the specific pol ζ REV3 S01, AS01, AS02, AS03, or SCRA control oligonucleotide, from day –1 to day 13. B cells were harvested at days 1, 2, 5, 7, 9, and 14 by positive selection with a biotinylated anti-CD19 mAb and MiniMac System Streptavidin microbeads (Miltenyi Biotec, Inc.) for RNA extraction. Shown are the transcript levels of pol ζ REV3, pol η, pol ι, pol α, pol β, and pol μ, as measured by RT-PCR after 9 days of cultures (comparable pol ζ inhibition was observed at all other time points analyzed). These findings were derived from one of three independent experiments yielding comparable results (data are from the experiments of Table 1). (B) Specific pol ζ REV3 oligonucleotides selectively inhibit the expression of pol ζ REV3 in CL-01 cells cultured in the absence of activated CD4+ T cells and without prior BCR engagement. All other experimental conditions and analyses are as in (A). (C and D) Frequency of the mutations in the Ig VHDJH and bcl-6 transcripts in the B cells from the cultures of (A). (E) CL-01 cells were treated with Cytofectin GSV only or Cytofectin GSV together with the pol ζ REV3 S01, AS01, SCRA, AS02, or AS03 oligonucleotides and then analyzed for the damage-induced mutagenesis using the UV-treated pSP189 shuttle vector. Histograms depict the frequency of 6-thioguanine-resistant mutants (white colonies out of about 4000 total colonies for each treatment).

Figure 5

Figure 5. Cytofectin GSV and the pol ζ REV3 Phosphorothioate Oligonucleotides Do Not Significantly Affect B Cell Cycle or Proliferation

(A) CD19+ cells from CL-01 cultures (same or similar to those of Figure 4) treated daily with Nil, Cytofectin GSV only, and Cytofectin GSV together with the SCRA, S01, or AS01 oligonucleotides, respectively, were harvested for counting and analysis of viability at days 0, 1, 2, 5, 7, 9, and 14 of culture. More than 90% of the B cells were viable at any time point. (B) B cell (from [A]) cycle analysis by PI staining at days 0, 2, 7, 9, and 14 of culture.

Similar articles

Cited by

References

    1. Aoufouchi S, Flatter E, Dahan A, Faili A, Bertocci B, Storck S, Delbos F, Cocea L, Gupta N, Weill JC, Reynaud CA. Two novel human and mouse DNA polymerases of the pol X family. Nucleic Acids Res. 2000;28:3684–3693. - PMC - PubMed
    1. Bachl J, Wabl M. Hypermutation in T cells questioned. Nature. 1995;375:285–286. - PubMed
    1. Bebenek K, Matsuda T, Masutani C, Hanaoka F, Kunkel TA. Proofreading of DNA polymerase η-dependent replication errors. J. Biol. Chem. 2001;276:2317–2320. - PubMed
    1. Bemark M, Khamlichi AA, Davies SL, Neuberger MS. Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Curr. Biol. 2000;10:1213–1216. - PubMed
    1. Bertocci B, Quint L, Delbos F, Garcia C, Reynaud CA, Weill JC. Probing immunoglobulin gene hypermutation with microsatellites suggests a nonreplicative short patch DNA synthesis process. Immunity. 1998;9:257–265. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources