REV1 Human

What is human REV1 and what is its primary function?

Human REV1 is a specialized DNA polymerase that functions primarily as a deoxycytidyl monophosphate (dCMP) transferase. It specifically inserts dCMP residues opposite DNA template guanine (G) bases and various DNA lesions. The human REV1 gene encodes a protein of 1251 amino acid residues with a calculated molecular weight of 138,248 Da. REV1 is an essential component of the DNA polymerase ζ mutagenesis pathway, playing a critical role in translesion DNA synthesis (TLS) to bypass lesions that would otherwise block replication fork progression .

Beyond its enzymatic activity, REV1 serves as a crucial scaffolding protein that tethers error-prone polymerases to stalled DNA replication forks. This structural function appears to be independent of its polymerase activity but integral to mutagenic bypass mechanisms .

How is the human REV1 gene organized and expressed?

The human REV1 gene is localized between chromosomal regions 2q11.1 and 2q11.2. Consistent with its fundamental role in DNA damage response, REV1 is ubiquitously expressed across various human tissues . This universal expression pattern supports its function as a fundamental mutagenic protein involved in translesion synthesis pathways.

The mutagenesis pathway involving REV1 appears to be evolutionarily conserved from yeast to humans, with homologs of yeast RAD6, RAD18, REV1, REV3, and REV7 all identified and cloned in human cells . This conservation underscores the fundamental importance of this pathway in cellular responses to DNA damage.

What are the unique enzymatic characteristics of human REV1?

Human REV1 exhibits several distinctive enzymatic properties that differentiate it from other DNA polymerases:

• It functions primarily as a dCMP transferase, preferentially inserting cytosine regardless of the template base .

• It lacks 3'→5' proofreading exonuclease activity, consistent with its role in translesion synthesis where accuracy may be compromised to facilitate replication past DNA damage .

• It shows a strong preference for template G but can operate on other template bases with reduced efficiency .

• It synthesizes DNA in a distributive manner rather than a processive one, as evidenced by the generation of products differing in length by single nucleotides .

Table 1: Relative Efficiency of dCMP Insertion by Human REV1 Opposite Different Template Bases

How does human REV1 respond to different DNA template bases and lesions?

Human REV1 demonstrates remarkable versatility in its response to various DNA template bases and lesions:

• For undamaged templates, REV1 inserts dCMP with highest efficiency opposite template G, followed by template A (6-fold lower efficiency), and then templates T and C (19-27-fold lower efficiency) .

• Regardless of the template base, REV1 predominantly inserts dCMP, showing a strong preference for this nucleotide .

• REV1 efficiently and specifically inserts dCMP opposite DNA lesions including apurinic/apyrimidinic (AP) sites and uracil residues .

• It can also insert dCMP opposite oxidative lesions such as 8-oxoguanine and other DNA adducts .

• For certain lesions like 6-ethenoadenine, REV1 participates in a "two-polymerase two-step mechanism" working in concert with other specialized polymerases .

This ability to respond to diverse types of DNA damage by consistently inserting dCMP makes REV1 a critical player in translesion synthesis across a wide spectrum of DNA lesions.

How does the structure of human REV1 compare to its yeast homolog?

The structure of human REV1 reveals significant differences from its yeast counterpart despite conservation of core catalytic functions:

• The human REV1 catalytic core is substantially larger than yeast REV1, with only 27% sequence identity between the two proteins .

• Human REV1 contains several unique structural elements, including large insertion regions that are absent in yeast REV1 .

• While both proteins mediate template G and incoming dCTP pairing via polymerase segments rather than direct base-pairing, the palm and fingers domains show substantial structural differences .

Table 2: Structural Comparison Between Human and Yeast REV1

What functional roles do the unique structural elements of human REV1 serve?

The distinctive structural elements of human REV1 appear to serve important functional roles:

• The palm domain contains a large 38-residue insert (I1) that extends more than 20Å away from the active site. Only the first 14 residues of this insert are visible in the electron density, suggesting the remaining portion becomes ordered only upon association with other proteins .

• The fingers domain features an insert (I2) that acts as a "flap" on the hydrophobic pocket accommodating template G, potentially influencing template base recognition .

• These unique structural elements, particularly I1, may provide additional interfaces for protein-protein interactions specific to human cells, reflecting the more complex requirements for coordinating translesion synthesis in higher eukaryotes .

• The structural arrangements facilitate REV1's role as a "hub" for protein-protein interactions in translesion DNA synthesis, allowing it to recruit and coordinate various specialized polymerases at sites of DNA damage .

How does REV1 contribute to translesion DNA synthesis pathways?

REV1 plays dual roles in translesion DNA synthesis pathways:

• As a specialized DNA polymerase, REV1 can directly participate in lesion bypass by inserting dCMP opposite various DNA lesions, including AP sites, uracil residues, and certain DNA adducts .

• As a scaffolding protein, REV1 serves to tether error-prone polymerases to sites of stalled DNA replication forks, facilitating the recruitment and coordination of other specialized polymerases .

• The scaffolding function appears to be more critical for mutagenesis than the catalytic activity, as evidenced by the observation that REV1 mutants that retain polymerase activity can be deficient in mutagenesis .

• REV1 interacts with homologous Y-family DNA polymerases and the REV7 subunit of polymerase ζ, creating a network of protein-protein interactions that coordinates the complex process of translesion synthesis .

What is the mechanism of REV1-mediated nucleotide insertion?

The mechanism of REV1-mediated nucleotide insertion differs fundamentally from that of conventional DNA polymerases:

• While most DNA polymerases facilitate direct base-pairing between the incoming nucleotide and the template base, REV1 mediates the pairing of template G and incoming dCTP with segments of the polymerase itself .

• This unique mechanism explains REV1's strong preference for inserting dCMP regardless of the template base, as the selection is determined more by protein-nucleotide interactions than by traditional Watson-Crick base-pairing .

• The "flap" created by the I2 insert in the fingers domain influences the hydrophobic pocket that accommodates template G, potentially contributing to REV1's base preference .

• This protein-directed mechanism allows REV1 to maintain consistent dCMP insertion even opposite damaged bases where normal base-pairing would be disrupted .

What evidence links REV1 to carcinogenesis?

Several lines of evidence connect REV1 to carcinogenesis:

• REV1 is directly implicated in the development of carcinogen-induced lung cancer, suggesting a role in the mutagenic processing of carcinogen-induced DNA adducts .

• As a key component of the mutagenic translesion DNA synthesis pathway, REV1 contributes to the generation of mutations following exposure to various mutagens .

• The ubiquitous expression of REV1 across human tissues correlates with its potential involvement in mutagenesis and carcinogenesis in multiple tissue types .

• Reduced expression of REV1 in cultured human cells results in significant reduction of UV-induced mutagenesis, demonstrating its role in generating mutations following DNA damage .

What experimental approaches are used to study REV1's role in cancer development?

Researchers employ various experimental approaches to investigate REV1's role in cancer development:

• Biochemical assays to characterize REV1's enzymatic activities, including DNA polymerase assays, kinetic analysis of nucleotide insertion, and studies of REV1's activity on different DNA templates and lesions .

• Structural studies using X-ray crystallography to determine the three-dimensional structure of human REV1 in complex with DNA and incoming nucleotides .

• Genetic engineering techniques, such as the design of hammerhead ribozymes targeting REV1 mRNA to reduce its expression levels. These ribozymes are designed based on predicted accessible loop structures of REV1 mRNA .

• Comparative studies between human and mouse REV1 (86% sequence identity), allowing the use of mouse models to investigate REV1's role in carcinogenesis .

• Cellular studies examining the effects of reduced REV1 expression on mutagenesis and sensitivity to carcinogens, supporting the hypothesis that lowering REV1 levels can alter mutagenic responses to carcinogen exposure .

Table 3: Response of Human REV1 to Different DNA Lesions