Somatic mutations to arginine (R) are a common feature of a subset of
J558 H chain genes that code for the majority of high-affinity, anti-d
sDNA antibodies in autoimmune MRL/lpr mice. To examine the consequence
s of such amino acid substitutions on DNA binding, we reverted three s
omatic mutations of a prototypic anti-dsDNA H chain gene, V(H)3H9, and
assayed the effect of those reversions by expression in a V(lambda)1
L chain-only plasmacytoma line. Reversion of R53 eliminated virtually
all dsDNA binding and sharply reduced ssDNA affinity. While the comple
te germ-line revertant of V(H)3H9 retained a low level of DNA binding,
the substitution of R96, a product of N base addition in the third co
mplementarity determining region (CDR3), with glycine (G) was sufficie
nt to abolish measureable DNA specificity. Antibodies with higher affi
nity for DNA were generated by introducing arginines into V(H)3H9 at a
ny one of four positions where somatic mutations to arginine had been
identified by sequencing other anti-dsDNA J558 H chain genes. All four
arginine mutants showed affinity increments consistent with their dir
ect involvement in DNA binding, although one such mutant, K64R, requir
ed the simultaneous reversion of an adjacent aspartic acid (D) to the
germ-line glycine. Two variants with three nongerm-line arginines show
ed further improvements in DNA affinity suggesting that their contribu
tions to DNA binding may be additive. Molecular modeling of antibody a
nd mutant F(ab) structures and calculations of their electrostatic pot
entials were used as an aid in interpreting the results and in predict
ing the location and size of possible combining sites.