requires an alternative pathway that is med-
iated by the 3′exonuclease activities of Pold,
which removes nucleotides from the 3′end of
an upstream Okazaki fragment, generating a
gap for the unprocessed 5′flap to reanneal for
ligation ( 16 , 23 ). Restrictive temperature stress
activates Dun1 signaling and stimulates de
novo production of deoxyribonucleotides, which
in turn inhibits the 3′exonuclease activity, but
not the flap nuclease activity of Pold, and in-
duces other DNA damage responses. These
molecular changes push OFM toward trans-
formation of an unprocessed 5′flap into a 3′
flap, either through flap equilibration ( 24 ) or
through the actions of helicases such as Sgs1 or
Pif1, leading to a secondary structure that may
result in alternative duplications, including
Pold-ITD, in revertant strains. In the rever-
tants, Poldmutations limit DNA displacement,
thus suppressing 5′flap formation or allowing
moretimeforDna2orExo1toactonthe5′
flap and bypass the requirement for Rad27
(Fig. 4G).
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ACKNOWLEDGMENTS
We thank R. Kolodner for the yeast strains RDKY2672, RDKY2608,
and RDKY2669; P. M. J. Burgers for the plasmids pBL335 (GST-
Pol3), pBL338 (GAL1-Pol31), pBL340 (GAL10-Pol32), and pBL341
(Pol31/Pol32); L. Prakash and S. Prakash for the protease-deficient
yeast strain YRP654 and the plasmids pBJ1445 (Flag-Pol3) and
pBJ1524 (GST-Pol31/Pol32) to express the yeast recombinant Pold
complex (Pol3, Pol31, and Pol32); W.-D. Heyer for the anti-Dun1
antibody; and M. S. Wold for purified recombinant yeast replication
protein A (RPA) complex. We thank H. Lou’s laboratory members
and H. Dai, D. Duenas, and M. E. Budd for technical assistance in
mouse and yeast genetic experiments and stimulating discussions.
We thank K. Walker and S. Wilkinson for critical reading and editing
of the manuscript.Funding:This work was supported by NIH
grants R50 CA211397 to L.Z. and R01 CA073764 and R01
CA085344 to B.S. Research reported in this publication includes
work performed by City of Hope shared resources supported by
the National Cancer Institute of the NIH under award number P30
CA033572.Author contributions:H.S., Z.L., A.S., Y.Z., and M.Z.
conducted yeast genetic and biochemical experiments. E.Z., J.W.,
X.W., Z.H., and Z.G. conducted RNA sequencing (RNA-seq), WES,
and WGS and performed data analysis. J.L.C. designed yeast
genetic experiments and conducted data analysis. L.Z. conducted
biochemical experiments, RNA-seq, and WGS data analysis;
designed and coordinated most of the experiments; and wrote the
first draft of the manuscript. B.S. supervised the entire project;
designed and coordinated most of the experiments; and providedinput into and finalized the manuscript.Competing interests:The
authors declare no conflicts of interest in this study.Data and
materials availability:All data are available in the manuscript or
the supplementary materials. Gene Expression Omnibus accession
numbers for the mouse and yeast genomics datasets are
GSE181154 and GSE178876, respectively.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abj1013
Materials and Methods
Supplementary Text
Figs. S1 to S20
Tables S1 to S6
References ( 25 Ð 51 )
MDAR Reproducibility Checklist20 April 2021; accepted 14 October 2021
10.1126/science.abj1013ORGANIC CHEMISTRYA biomimetic SH2 cross-coupling mechanism for
quaternary sp
3
-carbon formationWei Liu^1 †, Marissa N. Lavagnino^1 †, Colin A. Gould^1 , Jesús Alcázar^2 , David W. C. MacMillan^1 *Bimolecular homolytic substitution (SH2) is an open-shell mechanism that is implicated across
a host of biochemical alkylation pathways. Surprisingly, however, this radical substitution
manifold has not been generally deployed as a design element in synthetic C–C bond formation. We
found that the SH2 mechanism can be leveraged to enable a biomimetic sp^3 -sp^3 cross-coupling
platform that furnishes quaternary sp^3 -carbon centers, a long-standing challenge in organic
molecule construction. This heteroselective radical-radical coupling uses the capacity of iron
porphyrin to readily distinguish between the SH2 bond-forming roles of open-shell primary and
tertiary carbons, combined with photocatalysis to generate both radical classes simultaneously
from widely abundant functional groups. Mechanistic studies confirm the intermediacy of a primary
alkyl–Fe(III) species prior to coupling and provide evidence for the SH2 displacement pathway
in the critical quaternary sp^3 -carbon bond formation step.O
ver the past five decades, transition
metal–catalyzed cross-coupling has com-
prehensively transformed the landscape
of molecule construction in the applied
sciences, especially with respect to phar-
maceuticals, agrochemicals, and functional
materials ( 1 , 2 ). In particular, the combination
of three mechanistic steps—oxidative addition,
transmetalation, and reductive elimination—
has served as a robust catalytic paradigm for
C–C bond formation, enabling a highly mod-
ular yet general approach to fragment cou-
pling (Fig. 1A). Although this paradigm has
proven to be exceptionally successful for
forging C(sp^2 )–C(sp^2 ) bonds, it is important to
recognize that each of these three elementary
steps is less efficient when transition metals
engage with secondary or tertiary alkyl frag-
ments, limiting the development of a C(sp^3 )–C(sp^3 ) cross-coupling platform of broad util-
ity ( 3 – 6 ).
It is intriguing to consider that enzymatic
formation of C(sp^3 )–C(sp^3 ) bonds proceeds by
fundamentally different open-shell pathways
to achieve pivotal alkylation reactions ( 7 , 8 ).
As one canonical example, methylcobalamin
systems serve as nature’s“free radical carrier”
by stabilizing otherwise highly reactive methyl
radicals ( 9 , 10 ). As such, in cobalamin-dependent
radicalS-adenosylmethionine (SAM) meth-
yltransferases, transiently generated carbon-
centered radicals can react with these alkyl-
cobalt complexes via bimolecular homolytic
substitution (SH2) (Fig. 1B) ( 11 ). Although
cobalamin provides critical stabilization to
the reactive methyl radical, the methyl-cobalt
bond remains notably weak (bond dissocia-
tion energy = ~37 kcal/mol), which underpins
the kinetic preference for the SH2 mechanism
and heteroselective carbon-carbon bond for-
mation ( 12 ). Elegant biosynthetic studies have
shown that the rates of such enzymatic SH 2
reactions are extremely fast (~ 10^8 s–^1 ) and
enable the formation of sterically congested1258 3 DECEMBER 2021•VOL 374 ISSUE 6572 science.orgSCIENCE
(^1) Merck Center for Catalysis at Princeton University,
Princeton, NJ 08544, USA.^2 Discovery Chemistry, Janssen
Research and Development, Janssen-Cilag S.A., C/Jarama
75A, Toledo 45007, Spain.
*Corresponding author. Email: [email protected]
†These authors contributed equally to this work.
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