Requirement of DNA Polymerase η for Error-Free Bypass of UV-Induced CC and TC Photoproducts

ABSTRACT The yeast RAD30-encoded DNA polymerase η (Polη) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase η functions similarly in the bypass of this lesion, and mutations in human Polη result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation ofcis-syn cyclobutane dimers and (6-4) photoproducts at 5′-CC-3′ and 5′-TC-3′ sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3′ C to T transitions. Genetic studies presented here reveal a role for yeast Polη in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Polη plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.

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Role of DNA Polymerase η in the Bypass of a (6-4) TT Photoproduct

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3558-3563.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3558-3563 ◽

Cited By ~ 142

Author(s):

Robert E. Johnson ◽

Lajos Haracska ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Dna Polymerase ◽

Xeroderma Pigmentosum ◽

Uv Light ◽

Dna Lesions ◽

Variant Form ◽

Replication Machinery ◽

Genetic Studies ◽

Induced Mutagenesis ◽

Combined Action

ABSTRACT UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase η (Polη), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Polη result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Polη for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than acis-syn TT dimer. Opposite the 3′ T of a (6-4) TT photoproduct, both yeast and human Polη preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polζ, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3′ T of the (6-4) TT lesion by Polη, and Polζ inserts the correct nucleotide A opposite the 5′ T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Polη and Polζ, wherein Polη inserts a nucleotide opposite the 3′ T of the lesion and Polζ extends from it. These biochemical observations are in concert with genetic studies in yeast indicating that mutations occur predominantly at the 3′ T of the (6-4) TT photoproduct and that these mutations frequently exhibit a 3′ T→C change that would result from the insertion of a G opposite the 3′ T of the (6-4) TT lesion.

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Replication pastO6-Methylguanine by Yeast and Human DNA Polymerase η

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8001-8007.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8001-8007 ◽

Cited By ~ 85

Author(s):

Lajos Haracska ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Brain Tumors ◽

Dna Polymerase ◽

Double Mutant ◽

Malignant Tumors ◽

Alkylating Agents ◽

Dna Polymerases ◽

Clinical Effectiveness ◽

Induced Mutations ◽

Human Dna ◽

Biochemical Studies

ABSTRACT O 6-Methylguanine (m6G) is formed by the action of alkylating agents such asN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase η (Polη) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in therad30Δ pol32Δ double mutant of yeast, which lacks theRAD30 gene that encodes Polη and the Pol32 subunit of DNA polymerase δ (Polδ). Although Polδ can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Polη is much more efficient in replicating through m6G than Polδ. Both Polη and Polδ insert a C or a T residue opposite from m6G; Polη, however, is more accurate, as it inserts a C about twice as frequently as Polδ. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Polη could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.

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Development of an Automated UV Irradiation Device for Microbial Cell Culture

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630318800283 ◽

2018 ◽

Vol 24 (3) ◽

pp. 342-348

Author(s):

Atsushi Shibai ◽

Saburo Tsuru ◽

Tetsuya Yomo

Keyword(s):

Cell Culture ◽

Uv Irradiation ◽

Uv Light ◽

Accumulation Rate ◽

Microbial Cell ◽

Automated System ◽

Induced Mutations ◽

Uv Mutagenesis ◽

High Accumulation ◽

Irradiation Device

Ultraviolet (UV) mutagenesis is a widely used technique to increase bacterial mutation rates in laboratory experiments. UV mutagenesis requires fine regulation of UV dose, because the number of dead cells increases exponentially as the dose increases. Ignoring this hazard can cause extinction of UV-exposed populations. Therefore, an automated system that cooperatively conducts both growth measurement and UV irradiation is needed for efficient UV mutagenesis experiments. To address this task, we constructed an automated UV irradiation device for microbial cell culture. This device can measure cell density and irradiate the bacterial cells with UV light automatically according to the state of cell growth. We demonstrated that this growth feedback control avoided extinction and enabled accumulation of mutations in bacterial genomes at a rapid rate for a long period. Whole-genome sequencing revealed the high accumulation rate, neutrality, and spectrum of UV-induced mutations. These characteristics were all consistent with those obtained by manual UV irradiation. These results indicate that our automated device is useful in accelerating mutation accumulation over a long duration.

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Physical and Functional Interactions of Human DNA Polymerase η with PCNA

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7199-7206.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7199-7206 ◽

Cited By ~ 178

Author(s):

Lajos Haracska ◽

Robert E. Johnson ◽

Ildiko Unk ◽

Barbara Phillips ◽

Jerard Hurwitz ◽

...

Keyword(s):

Dna Polymerase ◽

Protein A ◽

Dna Lesions ◽

Nuclear Antigen ◽

Synthetic Activity ◽

Binding Motif ◽

Variant Form ◽

Physical Interaction ◽

Human Dna ◽

Factor C

ABSTRACT Human DNA polymerase η (hPolη) functions in the error-free replication of UV-damaged DNA, and mutations in hPolη cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPolη is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPolη with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPolη inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPolη, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparentK m for the incoming nucleotide.

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Yeast DNA Polymerase ζ Is an Efficient Extender of Primer Ends Opposite from 7,8-Dihydro-8-Oxoguanine and O6-Methylguanine

Molecular and Cellular Biology ◽

10.1128/mcb.23.4.1453-1459.2003 ◽

2003 ◽

Vol 23 (4) ◽

pp. 1453-1459 ◽

Cited By ~ 78

Author(s):

Lajos Haracska ◽

Satya Prakash ◽

Louise Prakash

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Uv Light ◽

Dna Lesions ◽

Geometric Distortion ◽

Cyclobutane Pyrimidine Dimers ◽

Genetic Studies ◽

Abasic Sites ◽

Dna Damaging Agents ◽

Pyrimidine Dimers

ABSTRACT Genetic studies in Saccharomyces cerevisiae have indicated the requirement of DNA polymerase (Pol) ζ for mutagenesis induced by UV light and by other DNA damaging agents. However, on its own, Polζ is highly inefficient at replicating through DNA lesions; rather, it promotes their mutagenic bypass by extending from the nucleotide inserted opposite the lesion by another DNA polymerase. So far, such a role for Polζ has been established for cyclobutane pyrimidine dimers, (6-4) dipyrimidine photoproducts, and abasic sites. Here, we examine whether Polζ can replicate through the 7,8-dihydro-8-oxoguanine (8-oxoG) and O 6-methylguanine (m6G) lesions. We chose these two lesions for this study because the replicative polymerase, Polδ, can replicate through them, albeit weakly. We found that Polζ is very inefficient at inserting nucleotides opposite both these lesions, but it can efficiently extend from the nucleotides inserted opposite them by Polδ. Also, the most efficient bypass of 8-oxoG and m6G lesions occurs when Polδ is combined with Polζ, indicating a role for Polζ in extending from the nucleotides inserted opposite these lesions by Polδ. Thus, Polζ is a highly specialized polymerase that can proficiently extend from the primer ends opposite DNA lesions, irrespective of their degree of geometric distortion. Polζ, however, is unusually sensitive to geometric distortion of the templating residue, as it is highly inefficient at incorporating nucleotides even opposite the moderately distorting 8-oxoG and m6G lesions.

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