Lai Man Chi

Effect of 1-methyladenine on double-helical DNA structures

Methylation at the N1 site of adenine leads to the formation of cytotoxic 1‐methyladenine (m1A). Since the N1 site of adenine is involved in the hydrogen bonding of T·A and A·T Watson–Crick base pairs, it is expected that the pairing interactions will be disrupted upon 1‐methylation. In this study, high‐resolution NMR investigations were performed to determine the effect of m1A on double‐helical DNA structures. Interestingly, instead of disrupting hydrogen bonding, we found that 1‐methylation altered the T·A Watson–Crick base pair to T(anti)·m1A(syn) Hoogsteen base pair, providing insights into the observed differences in AlkB‐repair efficiency between dsDNA and ssDNA.

The origin of genetic instability in CCTG repeats

CCTG tetranucleotide repeat expansion is associated with a hereditary neurological disease called myotonic dystrophy type 2 (DM2). The underlying reasons that lead to genetic instability and thus repeat expansion during DNA replication remains elusive. Here, we have shown CCTG repeats have a high propensity to form metastable hairpin and dumbbell structures using high-resolution nuclear magnetic resonance (NMR) spectroscopy. When the repeat length is equal to three, a hairpin with a two-residue CT loop is formed. In addition to the hairpin, a dumbbell structure with two CT-loops is formed when the repeat length is equal to four. Nuclear Overhauser effect (NOE) and chemical shift data reveal both the hairpin and dumbbell structures contain a flexible stem comprising a C-bulge and a T·T mismatch. With the aid of single-site mutation samples, NMR results show these peculiar structures undergo dynamic conformational exchange. In addition to the intrinsic flexibility in the stem region of these structures, the exchange process also serves as an origin of genetic instability that leads to repeat expansion during DNA replication. The structural features provide important drug target information for developing therapeutics to inhibit the expansion process and thus the onset of DM2.

NMR investigation of DNA primer–template models: Structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer–templates, NMR structural investigation has been performed on primer–template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.

Sequence Context Effect on Strand Slippage in Natural DNA Primer–Templates

Strand slippage has been found to occur in primer-templates containing a templating thymine, cytosine, and guanine, leading to the formation of misaligned structures with a single-nucleotide bulge. If remained in the active site of low-fidelity polymerases during DNA replication, these misaligned structures can ultimately bring about deletion mutations. In this study, we performed NMR investigations on primer-template models containing a templating adenine. Similar to our previous results on guanine, adenine templates are also less prone to strand slippage than pyrimidine templates. Misalignment occurs only in primer-templates that form a terminal C·G or G·C base pair. Together with our previous findings on thymine, cytosine, and guanine templates, the present study reveals strand slippage can occur in any kind of natural templating bases during DNA replication, providing insights into the origin of mutation hotspots in natural DNA sequences. In addition to the type of incoming base upon misincorporation, the propensity of strand slippage in primer-templates depends also on the type of templating base, its upstream and downstream bases.

Effect of hyperoxidized guanine on DNA primer–template structures: Spiroiminodihydantoin leads to strand slippage

Oxidation of guanine in DNA can lead to mutagenic lesions such as 7‐hydro‐8‐oxoguanine (oG). Upon further oxidation, a more mutagenic lesion, spirominodihydantoin (Sp), can occur. In this study, nuclear magnetic resonance (NMR) investigations were performed to determine the structural features of DNA primer–template models with 5′‐GG, 5′‐G(oG), 5′‐G(Sp) and 5′‐T(Sp) templates, that mimic the situation in which the downstream G of the template has been oxidized to oG or hyperoxidized to Sp. Our results show that misalignment occurs only in the 5′‐G(Sp) and 5′‐T(Sp) templates, providing structural insights into the observed differences in mutagenicity of Sp and oG during DNA replication.