in vivo

Recent publications
Masuda, K., Ouchida, R., Hikida,
M., Kurosaki, T., Yokoi, M.,
Masutani, C., Seki, M., Wood, R.
D., Hanaoka, F., O-Wang, J. DNA
polymerases η and θ function
in the same genetic pathway to
generate mutations at A/T during
somatic hypermutation of Ig genes.
J. Biol. Chem. 282: 17387-17394
(2007) .
Masuda, K., Ouchida, R., Hikida,
M., Nakayama, M., Ohara, O.,
Kurusaki, T., and O-Wang, J.
Absence of DNA polymerase
θ results in decreased somatic
hypermutation frequency and
altered mutation patterns in Ig
genes. DNA Repair 5: 1384-1391
(2006).
Figure
GC B cells have an intrisic property to generate A:T mutations in Ig and non-Ig genes. In Ig genes, AID
deaminates cytosine (C) to uracil (U) and generates a U:G lesion. Under normal situations, this U:G lesion is
correctly repaired by the error-free base excision repair (BER) pathway. During Ig gene SHM, however, the
intermidiates of the BER are resolved by error-prone repair pathways, leading to the mutations at nondamaged
A:T pairs. Using a lacZ-trangenic system, we found that GC B cells also efficiently generate A:T
mutations in non-Ig genes in an AID-independent but POLH-dependent manner.
A:T mutations observed in the absence of
POLH are generated by POLQ and how these
two polymerases might cooperate or compete
with each other to generate A:T mutations, we
have established and analyzed mice deficient
for both POLH and POLQ. Polq -/- Polh -/- mice,
however, did not show a further decrease of
A:T mutations as compared to Polh -/- mice,
suggesting that POLH and POLQ function in
the same genetic pathway in the generation of
these mutations. Frequent misincorporation of
nucleotides, in particular opposite a template T,
is a known feature of POLH, but the efficiency
of extension beyond the misincorporation differs
significantly depending on the nature of the
mispairing. Remarkably, we found that POLQ
catalyzed extension more efficiently than POLH
from all types of mispaired termini opposite
A or T. Moreover, POLQ was able to extend
mispaired termini generated by POLH albeit
at relatively low efficiency. These results reveal
genetic and biochemical interactions between
POLH and POLQ and suggest that POLQ might
cooperate with POLH to generate some of the
A:T mutations during SHM of Ig genes.
Mechanism of increased A:T mutations in
GC B cells
To understand why AID-triggered U:G lesions
lead to mutations at non-damaged A:T pairs in
GC B cells, we decided to compare mutation
patterns in non-Ig genes in GC B cells with those
in other cells/tissues. For this purpose, we utilized
a sensitive lacZ-transgenic (lacZ-Tg) system in
which genome mutations can be detected in an
unbiased way since there is no selective pressure
to obscure the lacZ mutations. We found that
approximately half of the mutations in the lacZ
gene occurred at A:T pairs in GC B cells, which
was in striking contrast to naïve B and non-GC
B cells where mutations occurred predominantly
at C:G pairs. To examine whether the increase of
A:T mutations is a specific feature of GC B cells,
we further analyzed mutation patterns in different
tissues. Mutations occurred exclusively at C:G
pairs in the brain, heart and liver, and only a small
fraction of the mutations occurred at A:T in the
small intestine. Importantly, the increased A:T
mutations in GC B cells were also observed in
mice lacking AID, indicating that the induction of
A:T mutations is a process independent of AIDmediated
Ig gene SHM. These results suggest
that GC B cells have an intrinsic propensity to
mutate A:T pairs in both Ig and non-Ig genes
(Figure). The GC B cells likely express a factor(s)
or provide a special environment that allows
efficient generation of A:T mutations not only
in resolving AID-triggered U:G lesions but also
during repair of endogenous DNA damage.
Maeda, Y., Fujimura, L., O-wang, J.,
Hatano, M., Sakamoto, A., Arima,
M., Ebara, M., Ino, H., Yamashita,
T., Saisho, H., and Tokuhiasa,T.
Role of Clast1 in development of
cerebellar granule cells. Brain Res.
1104:18-26 (2006).
Ukai, A., Maruyama, T., Mochizuki,
S., Ouchida, R., Masuda, K.,
Kawamura, K., Tagawa, M.,
Kinoshita, K., Sakamoto, A.,
Tokuhisa, T. and O-Wang, J. Role
of DNA polymerase θ in tolerance
of endogenous and exogenous
DNA damage in mouse B cells.
Genes Cells 11: 111-121 (2006).
Masuda, K., Ouchida, R., Takeuchi,
A., Saito, T., Koseki, H., Kawamura,
K., Tagawa, M., Tokuhisa, T.,
Azuma, T. and O-Wang, J. DNA
polymerase θ contributes to the
generation of C/G mutations during
somatic hypermutation of Ig genes.
Proc. Natl. Acad. Sci. USA 102:
13986-13991 (2005).
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