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DNA Polymerase V (Pol V) is a
polymerase A polymerase is an enzyme ( EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base- ...
enzyme involved in
DNA repair DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA dam ...
mechanisms in bacteria, such as ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
''. It is composed of a UmuD' homodimer and a UmuC
monomer In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization. Classification Mo ...
, forming the UmuD'2C protein complex. It is part of the Y-family of DNA Polymerases, which are capable of performing DNA translesion synthesis (TLS). Translesion polymerases bypass DNA damage lesions during
DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
- if a lesion is not repaired or bypassed the
replication fork In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
can stall and lead to cell death. However, Y polymerases have low sequence fidelity during replication (prone to add wrong nucleotides). When the UmuC and UmuD' proteins were initially discovered in ''E. coli'', they were thought to be agents that inhibit faithful DNA replication and caused DNA synthesis to have high mutation rates after exposure to
UV-light Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiation i ...
. The polymerase function of Pol V was not discovered until the late 1990s when UmuC was successfully extracted, consequent experiments unequivocally proved UmuD'2C is a polymerase. This finding lead to the detection of many Pol V orthologs and the discovery of the Y-family of polymerases.


Function

Pol V functions as a TLS (translesion DNA synthesis) polymerase in ''E. coli'' as part of the SOS response to DNA damage. When DNA is damaged regular DNA synthesis polymerases are unable to add dNTPs onto the newly synthesized strand.
DNA Polymerase III DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg (son of Arthur Kornberg) and Malcolm Gefter in 1970. The complex has high processivity (i.e. the number of ...
(Pol III) is the regular DNA polymerase in ''E. coli''. As Pol III stalls unable to add nucleotides to the nascent DNA strand, the cell becomes at risk of having the replication fork collapse and cell death to occur. Pol V TLS function depends on association with other elements of the SOS response, most importantly Pol V translesion activity is tightly dependent on the formation of RecA nucleoprotein filaments. Pol V can use TLS on lesions that block replication or miscoding lesions, which modify bases and lead to wrong
base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
ing. However, it is unable to translate through 5' → 3' backbone nick errors. Pol V also lacks
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...
activity, thus rendering unable to
proofread Proofreading is the reading of a galley proof or an electronic copy of a publication to find and correct reproduction errors of text or art. Proofreading is the final step in the editorial cycle before publication. Professional Traditional ...
synthesis causing it to be error prone.


SOS Response

SOS response in ''E. coli'' attempts to alleviate the effect of a damaging stress in the cell. The role of Pol V in SOS response triggered by UV-radiation is described as follows: # Pol III stalls at lesion site. # DNA replication helicase DnaB continues to expand the
replication fork In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
creating single stranded DNA (ssDNA) segments ahead of from the lesion. # ssDNA binding proteins (SSBs) stabilize ssDNA. # RecA recruited and loaded onto ssDNA by RecFOR replacing SSBs. Formation of RecA nucleoprotein filament (RecA*). # RecA functions through mediator proteins to activate Pol V (see Regulation). # Pol V accesses 3'-OH of nascent DNA strand and extends strand past the lesion site. # Pol III resumes elongation.


Regulation

Pol V is only expressed in the cell during the SOS response. It is very tightly regulated at different levels of protein expression and under different mechanisms to avoid its activity unless absolutely necessary for survival of the cell. Pol V's strict regulation stems from its poor replication fidelity, Pol V is highly mutagenic and it is used as a last resort in DNA repair mechanisms. As such, the expression of the UmuD'2C complex takes 45–50 minutes after UV radiation exposure.


Transcriptional regulation

Transcription of the SOS response genes is negatively regulated by the LexA repressor. LexA binds to the promoter of the UmuDC operon and inhibits gene transcription. DNA damage in the cell leads to the formation of RecA*. RecA* interacts with LexA and stimulates its proteolytic activity, which leads to the autocleavage of the repressor freeing the operon for transcription. The UmuDC operon is transcribed and translated into UmuC and UmuD.


Post-translational regulation

The formation of the UmuD'2C complex is limited by the formation of UmuD' from UmuD. UmuD is made of a polypeptide with 139 amino acid residues that form a stable tertiary structure, however it needs to be post-translationally modified to be in its active form. UmuD has self-proteolytic activity that is activated by RecA, it removes 24 amino acids at the
N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...
, turning it into UmuD'. UmuD' can form a homodimer and associate with UmuC to form the active UmuD'2C complex.


Functional regulation

UmuD'2C complex is inactive unless associated with RecA*. Pol V directly interacts with RecA* at the 3' tip of the nucleoprotein filament; this is the site of the nascent DNA strand where Pol V restarts DNA synthesis. Additionally, it has been shown that the
REV1 DNA repair protein REV1 is a protein that in humans is encoded by the ''REV1'' gene. This gene encodes a protein with similarity to the S. cerevisiae mutagenesis protein Rev1. The Rev1 proteins contain a BRCT domain, which is important in protei ...
/REV3L/REV7 pathway is necessary for the TLS synthesis mediated by DNA polymerase V.


References


External links

* {{Portal bar, Biology, border=no DNA replication EC 2.7.7 EC 3.4.21