Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday.

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

Kuvaus

Even though DNA alkylating agents have been used for many decades in treatment of cancer it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 hour of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks (SSBs). Overexpression of MGMT protein, responsible for removing the most toxic alkylation, O(6)-methyl guanine (O6meG), did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localize with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks (DSBs) are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely DSBs.
Alkuperäiskielienglanti
LehtiJournal of Molecular Biology
Vuosikerta402
Sivut70-82
ISSN0022-2836
TilaJulkaistu - 2010
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä, vertaisarvioitu

Tieteenalat

  • 1182 Biokemia, solu- ja molekyylibiologia

Lainaa tätä

01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday. (2010). Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage. Journal of Molecular Biology, 402, 70-82.
01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday. / Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage. Julkaisussa: Journal of Molecular Biology. 2010 ; Vuosikerta 402. Sivut 70-82.
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title = "Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage",
abstract = "Even though DNA alkylating agents have been used for many decades in treatment of cancer it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 hour of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks (SSBs). Overexpression of MGMT protein, responsible for removing the most toxic alkylation, O(6)-methyl guanine (O6meG), did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localize with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks (DSBs) are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely DSBs.",
keywords = "1182 Biochemistry, cell and molecular biology, DNA Damage Repair",
author = "Majumder, {Muntasir Mamun} and {01. Petra Groth, Simon Ausl{\"a}nder, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday.}",
year = "2010",
language = "English",
volume = "402",
pages = "70--82",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "ACADEMIC PRESS INC ELSEVIER SCIENCE",

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01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday. 2010, 'Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage', Journal of Molecular Biology, Vuosikerta 402, Sivut 70-82.

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage. / 01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday.

julkaisussa: Journal of Molecular Biology, Vuosikerta 402, 2010, s. 70-82.

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

TY - JOUR

T1 - Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

AU - Majumder, Muntasir Mamun

AU - 01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday.

PY - 2010

Y1 - 2010

N2 - Even though DNA alkylating agents have been used for many decades in treatment of cancer it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 hour of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks (SSBs). Overexpression of MGMT protein, responsible for removing the most toxic alkylation, O(6)-methyl guanine (O6meG), did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localize with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks (DSBs) are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely DSBs.

AB - Even though DNA alkylating agents have been used for many decades in treatment of cancer it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 hour of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks (SSBs). Overexpression of MGMT protein, responsible for removing the most toxic alkylation, O(6)-methyl guanine (O6meG), did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localize with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks (DSBs) are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely DSBs.

KW - 1182 Biochemistry, cell and molecular biology

KW - DNA Damage Repair

M3 - Article

VL - 402

SP - 70

EP - 82

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

ER -

01. Petra Groth, Simon Ausländer, Muntasir Mamun Majumder, Niklas Schultz, Fredrik Johansson, Eva Petermann, Thomas Helleday. Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage. Journal of Molecular Biology. 2010;402:70-82.