Sik Lok Lam

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.

DSHIFT: a web server for predicting DNA chemical shifts

DSHIFT is a web server for predicting chemical shifts of DNA sequences in random coil form or double helical B-form. The prediction methods are based on sets of published reference chemical shift values and correction factors which account for shielding or deshielding effects from neighboring nucleotides. Proton, carbon and phosphorus chemical shift predictions are available for random coil DNAs. For double helical B-DNA, only proton chemical shift prediction is available. Results from these predictions will be useful for facilitating NMR resonance assignments and investigating structural features of solution DNA molecules. The URL of this server is: http://www.chem.cuhk.edu.hk/DSHIFT.

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.

Effect of 1-methyladenine on thermodynamic stabilities of double-helical DNA structures

1‐Methyladenine (m1A) alters T·A Watson–Crick to T·m1A Hoogsteen base pair. Owing to its conversion to N6‐methyladenine (m6A) at higher temperatures, thermodynamic studies of m1A‐containing DNAs using conventional melting methods are subject to the influence of m6A species. In this study, we applied nuclear magnetic resonance spectroscopy to determine the base pairing modes and effect of m1A on thermodynamic stability of double‐helical DNA. The observed base pairing modes account for the destabilizing trend which follows the order T·m1A ∼ G·m1A < A·m1A < C·m1A, providing insights into the m1A flipping process and enhancing our understandings of the mutagenicity of m1A.

New insights into the genetic instability in CCTG repeats.

Tetranucleotide CCTG repeat expansion is associated with myotonic dystrophy type 2, which is an inherited and progressive muscle degeneration disease. Yet, no cure is available and the molecular mechanism of repeat expansion remains elusive. In this study, we used high‐resolution nuclear magnetic resonance spectroscopy to reveal a mini‐dumbbell structure formed by two CCTG repeats. Upon slippage in the nascent strand during DNA replication, the formation of the mini‐dumbbell provides a possible pathway for a two‐repeat expansion. In addition, fast exchange between two competing mini‐dumbbells among three repeats results in a mini‐loop structure that accounts for one‐repeat expansion. These mini‐dumbbell and mini‐loop intermediates can also co‐exist at multiple sites in CCTG repeats, leading to three or larger size repeat expansions.

Preferential base pairing modes of T·T mismatches

It has long been recognized that T·T mismatches can adopt two different modes of exchangeable wobble base pairs in which no preferential pairing mode has been observed. In this study, we have performed a systematic nuclear magnetic resonance (NMR) investigation to study the sequence context effect on the pairing modes of T·T mismatches. Our results reveal for the first time that preferential pairing mode does exist in T·T mismatches with specific type of flanking base pairs.

Unusual structures of TTTA repeats in icaC gene of Staphylococcus aureus

One and two TTTA repeat expansions have been found in the coding region of icaC gene of Staphylococcus aureus variants which influence the expression of IcaC protein and alter the phenotype. Yet, the mechanism of these small‐size TTTA repeat expansions remains unclear. In this study, we performed high‐resolution nuclear magnetic resonance spectroscopic studies on TTTA repeats. Our results show that a DNA sequence containing three TTTA repeats can fold into dumbbell structures with a 3′ or 5′‐overhang. Exchange of these dumbbells makes the sequence behave like a 2‐nt TT mini‐loop at 25 °C. The occurrence of these mini‐loop and dumbbell structures in the nascent strand during DNA replication provides possible mechanistic pathways which account for one and two repeat expansions.

Low Temperature Solution Structures and Base Pair Stacking of Double Helical d(CGTACG)2

Abstract Solution structures and base pair stacking of a self-complementary DNA hexamer d(CGTACG)2 have been studied at 5, 10 and 15 °C, respectively. The stacking interactions among the center base pair steps of the DNA duplex are found to improve when the terminal base pairs became less stable due to end fraying. A new structural quantity, the stacking sum (∑s), is introduced to indicate small changes in the stacking overlaps between base pairs. The improvements in the stacking overlaps to maintain the double helical conformation are probably the cause for the observed temperature dependent structural changes in double helical DNA molecule. A detailed analysis of the helical parameters, backbone torsion angles, base orientations and sugar conformations of these structures has been performed.

Minidumbbell: A New Form of Native DNA Structure

The non-B DNA structures formed by short tandem repeats on the nascent strand during DNA replication have been proposed to be the structural intermediates that lead to repeat expansion mutations. Tetranucleotide TTTA and CCTG repeat expansions have been known to cause reduction in biofilm formation in Staphylococcus aureus and myotonic dystrophy type 2 in human, respectively. In this study, we report the first three-dimensional minidumbbell (MDB) structure formed by natural DNA sequences containing two TTTA or CCTG repeats. The formation of MDB provides possible pathways for strand slippage to occur, which ultimately leads to repair escape and thus expansion mutations. Our result here shows that MDB is a highly compact structure composed of two type II loops. In addition to the typical stabilizing interactions in type II loops, MDB shows extensive stabilizing forces between the two loops, including two distinctive modes of interactions between the minor groove residues. The formation of MDB enriches the structural diversity of natural DNA sequences, reveals the importance of loop-loop interactions in unusual DNA structures, and provides insights into novel mechanistic pathways of DNA repeat expansion mutations.