Structural Insight into Translesion Synthesis by DNA Pol II

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Somatic hypermutation is programmed base substitutions in the variable regions of Ig genes for high-affinity antibody generation. Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T), have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase η (Pol η) is responsible for converting the WA motif to WG by misincorporating dGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol η substituting dGTP for dATP in four sequence contexts, TA, AA, GA, and CA, have been determined and compared. The T:dGTP wobble base pair is stabilized by Gln-38 and Arg-61, two uniquely conserved residues among Pol η. Weak base paring of the W (T:A or A:T) at the primer end and their distinct interactions with Pol η lead to misincorporation of G in the WA motif. Between two WA motifs, our kinetic and structural data indicate that A-to-G mutation occurs more readily in the TA context than AA. Finally, Pol η can extend the T:G mispair efficiently to complete the mutagenesis.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η

Zhao

Gregory

Biertumpfel

et al. 2013

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“…Because hTHG1 shares no significant sequence similarity with any known protein, it was unclear whether hTHG1 would have any structural homologs. Surprisingly, significant homology was found with guanylyl and adenylyl cyclases [βαββαβ motif of C 1a domain; Z score ¼ 8.0; Protein Data Bank (PDB) ID codes 2W01 (21) and 3E8A (22)] (23), along with the palm domain of several traditional polymerases including T7 DNA polymerase, a family A polymerase [ Z score ¼ 5.3, PDB ID code 1T7P (24)], and DNA polymerase II, a member of the B family [Z score ¼ 5.9, PDB ID code 1Q8I, (25)]. The fold of the hTHG1 βαββαβ motif (residues 22-135) most closely matches that of the cyclases.…”

Section: Resultsmentioning

confidence: 99%

tRNA ^His guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases

Hyde

Eckenroth

Smith

et al. 2010

131

All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5′ to 3′ direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA His guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3′-5′ addition of nucleotides to nucleic acid substrates. The 2.3-Å crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5′-3′ DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5′-3′ and 3′-5′ reactions raises important questions concerning selection of the 5′-3′ mechanism during the evolution of nucleotide polymerases. G-1 addition | reverse polymerase | tRNA modificationA ll nucleotide polymerases, including DNA and RNA polymerases, reverse transcriptase, and telomerase, catalyze nucleotide addition in the 5′ to 3′ direction. The reaction involves the nucleophilic attack of a polynucleotide terminal 3′-OH onto the α-phosphate of an incoming nucleotide, followed by release of the pyrophosphate moiety. Although the 5′ to 3′ direction has been adopted by all polymerases and transferases described to date, there is one notable exception: the enzyme tRNA His guanylyltransferase (Thg1). Thg1 catalyzes the highly unusual 3′-5′ addition of a single guanine to the 5′-end of tRNA His (1, 2). This reaction is an obligatory step in the maturation of this tRNA because the extra 5′ base, G −1 , constitutes a primary identity element for the aminoacyl-tRNA synthetase (HisRS) that attaches the amino acid histidine to the 3′-end of the tRNA (3-9). Thg1 is thus essential for maintaining the fidelity of protein synthesis. Consistent with the critical nature of the G −1 residue, THG1 is an essential gene in yeast and RNAi-mediated silencing of the Thg1 homolog in human cells results in severe cell-cycle progression and growth defects (2, 10, 11). Thg1 is widely conserved throughout eukarya, and Thg1 homologs are present in many archaea and bacteria.In eukarya, G −1 addition occurs opposite a universally conserved A 73 and thus is the result of a nontemplated 3′-5′ addition reaction. In addition, yeast Thg1 catalyzes a second reaction in vitro, extending tRNA substrates in the 3′-5′ direction in a template-directed manner driven by Watson-Crick pairing (12). Thg1 enzymes in archaea also catalyze template-dependent 3′-5′ addition, but do not catalyze nontemplated G −1 addition (13), suggesting that the templated 3′-5′ addition reaction likely represents an ancestral activity of the earliest Thg1 family members.The 3′-5′ addition of G −1 to tRNA His occurs via three chemical reactions, all catalyzed by Thg1 (2, 14) (Fig. 1). First, the 5′-monophosphorylated tRNA that results from RNase P cleavage of pre-tRNA His is activated using ATP, creating a 5...

“…For example, interactions between PCNA and DNAP from Pyrococcus furiousos appear to decrease the binding of primer strands at the exo site and stabilize the P/T DNA duplex within the polymerase, thereby favoring processive elongation (26). In addition, partitioning of the primer terminus between the polymerase and exonuclease active sites modulates translesion synthesis by E. coli DNA Pol II (27). Polymerase features that are involved in control of the occupancy of the exo site are beginning to be investigated by protein engineering (27).…”

Section: A B C D Ementioning

confidence: 99%

“…In addition, partitioning of the primer terminus between the polymerase and exonuclease active sites modulates translesion synthesis by E. coli DNA Pol II (27). Polymerase features that are involved in control of the occupancy of the exo site are beginning to be investigated by protein engineering (27). Not all DNA polymerases have an exo binding mode; repair polymerases in the X and Y families and certain A-family polymerases that perform translesion and mutagenic DNA synthesis lack proofreading exonuclease activity (28).…”

Section: A B C D Ementioning

confidence: 99%

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Datta

Johnson

Hippel

2010

Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3′-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3′-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.DNA editing | Klenow DNA polymerase | primer partitioning in DNAP active sites | low energy circular dichroism | base analogues 6-MI and 2-AP