Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • A 804598 The larger eukaryotic ligases such as LigI and

    2021-04-01

    The larger eukaryotic ligases, such as LigI and LigIV, also possess an additional N-terminal DNA-binding domain (DBD) that is required for efficient ligation (Fig. 1c) and enables these ligases to encircle DNA [17]. An equivalent helix–hairpin–helix domain is also present in the bacterial NAD-dependent ligases [13], [18]. The toroidal structure of LigI maintains the nicked DNA in a distorted conformation and locates the catalytic domain over the site of the nick prior to ligation [17]. Since DNA ligase IV is essential, the mutations identified in LIG4 syndrome are hypomorphic, that is, they confer residual activity. The analysis of the impact of such mutations is important to help evaluate the clinical impact and potentially to help direct patient care. Additionally, such mutations have the potential to provide novel insight into domains or motifs important for function. In one LIG4 syndrome patient an arginine was mutated to histidine within motif I (R278H) close to the active site lysine residue [19], [20]. More recently, another mutational change (G469E) was identified [5]. Although this mutation lies adjacent to a conserved residue (G468), it lies outside of the six core motifs and, when first identified, we questioned whether it would alter the activity of the enzyme [21]. Our initial studies demonstrated, however, that it was a mutational change that impacted upon function [5]. A sequence comparison between DNA ligases strongly suggests that residues 468–476 represent a further conserved motif present in eukaryotic DNA ligases, which we have designated motif Va (Fig. 1b). The recently reported structure of DNA ligase I also provides evidence that this region might be important for function (Pascal, 2004 #10951). Here, we have undertaken a mutational analysis of residues within motif Va to determine whether it was also important for function in DNA ligase IV. Mutational change of either residue G469 or G468 to glutamic A 804598 markedly reduced protein expression, adenylation and ligation activity. G468A and G469A mutations were better tolerated but were still impacting. Our findings provide biochemical evidence supporting the importance of a glycine at position G468, which is apparent from the DNA ligase I/DNA crystal structure and the high conservation of this residue. Additionally, we found that residues 470–476 do not affect either DNA binding or adenylation activity but significantly impair double-stranded ligation activity. We discuss a model that these residues act, along with another conserved structural motif, as a pincer to facilitate the conformational change of the DNA to enhance catalysis.
    Materials and methods
    Results
    Discussion Previously, we reported the identification of a mutation, G469E, in a LIG4 syndrome patient which severely impacts upon DNA ligase IV function [5]. Closer examination of the sequence in the vicinity of G469 revealed a motif, designated Va, that is well conserved between DNA ligases (Fig. 1b). Mutational analysis of residues within motif Va highlights an important role in ligation. G468 represents a highly conserved residue, as noted previously [21] which, we show, is required for adenylate complex formation. In contrast, residues 470, 473 and 476 which lie within the loop region encompassing motif Va are dispensable for adenylate complex formation and for DNA binding but are required for efficient DNA ligation. During the course of this work, the crystal structure of the Ligase I:DNA complex was reported [17], enabling us to directly evaluate the observed mutational effects at the molecular level. G468 is buried within the OBD at a position of close packing between β sheets (Fig. 7a). Glycine is an atypical amino acid lacking charge and a side-chain. It is likely that substitution of this residue by other amino acids would severely compromise the stability of the OBD. Indeed, substitution of a glutamic acid, which is charged and has a large side-chain resulted in a protein that appeared to be poorly expressed, likely due to impaired stability. Substitution of the slightly smaller and uncharged alanine was slightly better tolerated but protein expression was still low. The G468E mutant protein showed little functional activity either for adenylation or ligation activities most likely due to a significant impact on conformation. G469, which is slightly less buried in the OBD, also appears to be important for protein conformation. Significantly, G469A is better tolerated than other substitutions at the 468/469 residues and yields a protein with measurable residual adenylation activity. This is consistent with the less stringent conservation of this residue, and indeed, DNA ligase I has an alanine at this site. Analysis of G468A mutant complexes demonstrated that they have impaired affinity for ATP. Whilst, our analysis at non-saturating ATP concentrations served to expose the defect in the mutant complexes, it is possible that under physiological ATP concentration, the adenylation activity of the mutant could be nearly normal. It is likely that the G469A mutant complex responds similarly to ATP concentrations, potentially explaining the apparent in vitro impact of this mutation on adenylation but not ligation. Since all the mutants generated at G468/9 residues show impaired stability in insect cells, we cannot conclude that these residues directly participate in catalytic activity. Rather our findings suggest that impaired activity, including a reduced affinity for ATP, is conferred as a consequence of altered protein conformation. Together, this analysis suggests that G469E generates a protein with significant reduced conformational stability that impacts upon activity. Nonetheless it retains some residual activity providing an explanation for the viability but significant clinical features of the patient.