O6-Methylguanine induces altered proteins at the level of transcription in human cells
John A. Burns
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Kristian Dreij
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Laura Cartularo
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David A. Scicchitano
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Department of Biology, New York University, 1009 Silver Center
, 100 Washington Square East,
New York, NY 10003, USA
O6-Methylguanine (O6-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O6-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O6-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O6-alkylguanine-DNA alkyltransferase (AGT), which repairs O6-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O6-benzylguanine, 58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O6-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.
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Radiation and chemicals can give rise to reactive species
that damage DNA, producing strand breaks, abasic sites,
and altered bases, sugars and phosphate groups. These
structural modifications to the genetic material often
exert detrimental effects on replication and gene
expression, compromising DNAs role as the repository of
cellular information. DNA-dependent DNA polymerases
can stall at damaged sites in the template, giving rise to
collapsed replication forks, or synthesize incorrect
products as they progress past the lesions, causing
mutations in the daughter DNA (13). DNA-dependent RNA
polymerases can also stall at damaged bases in DNA or
bypass the lesions, resulting in altered transcripts in a
process called transcriptional mutagenesis (48). To
avert the deleterious effects of DNA damage, cells have
evolved an elaborate array of DNA repair pathways that
maintain and preserve DNA structure. But when genomic
maintenance is compromised due to aberrant DNA repair,
the accumulated damage and associated effects on
replication and transcription result in pathology that can
include cancer and developmental deficits (9,10).
Among the chemical agents that damage DNA are
those that alkylate the purines, pyrimidines and phosphate
groups. Methylating agents, such as the chemical
N-methyl-N-nitrosourea (MNU) or the endogenous
metabolite S-adenosylmethionine, produce two abundant
lesions, 7-methylguanine, which is relatively innocuous,
and 3-methyladenine, which is cytotoxic. Methylating
agents that tend to be highly carcinogenic, which
include MNU, also form significant amounts of
O6-methylguanine (O6-meG) (1114). Furthermore,
the O6-position of guanine is a target site for alkylation
by certain chemotherapeutic drugs, such as 1,3-bis
(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide
(15,16). BCNU adds a chloroethyl group to the
O6-position of guanine, which subsequently rearranges
and forms a cytotoxic interstrand DNA crosslink (17).
Temozolomide spontaneously degrades, giving rise to a
methyldiazonium ion that methylates DNA, including
the O6-position of guanine (18). In both cases, the
formation of the O6-alkylated guanine plays an important role
in the cytotoxic mechanism of these drugs.
O6-MeG is highly mutagenic, instructing DNA
polymerases to incorporate thymine instead of cytosine
opposite the lesion, resulting in GC to AT transitions
during replication (1921). O6-MeG is also cytotoxic
because it invokes DNA mismatch repair that results in
a cycle of futile attempts at clearing the lesion, ultimately
producing DNA strand breaks and inducing apoptosis
(22). The mutagenic and cytotoxic consequences of
O6-meG are ameliorated by the presence of the protein
O6-alkylguanineDNA alkyltransferase (AGT) (2325).
AGT is found in both prokaryotes and eukaryotes and
works in a similar way in each. The protein binds to
O6-meG, removes the methyl group and transfers it to a
cysteine located within the AGT active site, generating
S-methylcysteine in the protein and restoring guanine in
DNA. AGTs action is not enzymatic: Its activity cannot
be regenerated after it has repaired a single lesion. AGT
can also remove longer chain alkyl groups from the
O6-position of guanine; hence, it should come as no
surprise that AGT interferes with the chemotherapeutic
efficacy of agents that alkylate the O6-position of
guanine. In the case of BCNU, AGT repairs the
O6-chloroethylguanine intermediate, thwarting the
formation of the cytotoxic DNA interstrand crosslink (26). For
temozolomide, AGT repairs O6-meG, thus clearing a
cytotoxic DNA lesion that induces apoptosis (18,27).
In bioche (...truncated)