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Article ; Online: Multiple endocrine neoplasia type 1 (MEN1) as a cancer predisposition syndrome: clues into the mechanisms of MEN1-related carcinogenesis.

Busygina, Valeria / Bale, Allen E

The Yale journal of biology and medicine

2007 Volume 79, Issue 3-4, Page(s) 105–114

MeSH term(s)	Genetic Predisposition to Disease ; Humans ; Models, Biological ; Multiple Endocrine Neoplasia Type 1/diagnosis ; Multiple Endocrine Neoplasia Type 1/pathology ; Mutation ; Nervous System Neoplasms/etiology ; Precancerous Conditions/diagnosis ; Precancerous Conditions/pathology ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins/physiology ; Risk Factors
Chemical Substances	MEN1 protein, human ; Proto-Oncogene Proteins
Language	English
Publishing date	2007-10-17
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	200515-3
ISSN	1551-4056 ; 0044-0086
ISSN (online)	1551-4056
ISSN	0044-0086
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Article ; Online: Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

Batonick, Melissa / Holland, Erika G / Busygina, Valeria / Alderman, Dawn / Kay, Brian K / Weiner, Michael P / Kiss, Margaret M

New biotechnology

2016 Volume 33, Issue 5 Pt A, Page(s) 565–573

Abstract: Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical ... ...

Abstract	Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.
MeSH term(s)	Amino Acid Sequence ; Antibody Diversity ; Biotechnology ; Complementarity Determining Regions/genetics ; Escherichia coli/genetics ; High-Throughput Screening Assays ; Humans ; Peptide Library ; Single-Chain Antibodies/biosynthesis ; Single-Chain Antibodies/genetics
Chemical Substances	Complementarity Determining Regions ; Peptide Library ; Single-Chain Antibodies
Language	English
Publishing date	2016-09-25
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2400836-9
ISSN	1876-4347 ; 1871-6784
ISSN (online)	1876-4347
ISSN	1871-6784
DOI	10.1016/j.nbt.2015.11.005
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Article ; Online: Role of replication protein A in double holliday junction dissolution mediated by the BLM-Topo IIIα-RMI1-RMI2 protein complex.

Xue, Xiaoyu / Raynard, Steven / Busygina, Valeria / Singh, Akhilesh K / Sung, Patrick

The Journal of biological chemistry

2013 Volume 288, Issue 20, Page(s) 14221–14227

Abstract: The conserved BTR complex, composed of the Bloom's syndrome helicase (BLM), topoisomerase IIIα, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show ...

Abstract	The conserved BTR complex, composed of the Bloom's syndrome helicase (BLM), topoisomerase IIIα, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show enhancement of the BTR-mediated dHJ dissolution reaction by the heterotrimeric single-stranded DNA binding protein replication protein A (RPA). Our results suggest that RPA acts by sequestering a single-stranded DNA intermediate during dHJ dissolution. We provide evidence that RPA physically interacts with RMI1. The RPA interaction domain in RMI1 has been mapped, and RMI1 mutants impaired for RPA interaction have been generated. Examination of these mutants ascertains the significance of the RMI1-RPA interaction in dHJ dissolution. Our results thus implicate RPA as a cofactor of the BTR complex in dHJ dissolution.
MeSH term(s)	Amino Acid Sequence ; Carrier Proteins/metabolism ; DNA/genetics ; DNA Repair ; DNA Topoisomerases, Type I/metabolism ; DNA, Cruciform ; DNA-Binding Proteins/metabolism ; Humans ; Molecular Sequence Data ; Mutation ; Nuclear Proteins/metabolism ; Protein Binding ; RecQ Helicases/metabolism ; Replication Protein A/metabolism ; Saccharomyces cerevisiae/metabolism ; Sequence Homology, Amino Acid
Chemical Substances	Carrier Proteins ; DNA, Cruciform ; DNA-Binding Proteins ; Nuclear Proteins ; RMI1 protein, human ; RMI2 protein, human ; Replication Protein A ; DNA (9007-49-2) ; Bloom syndrome protein (EC 3.6.1.-) ; RecQ Helicases (EC 3.6.4.12) ; DNA Topoisomerases, Type I (EC 5.99.1.2)
Language	English
Publishing date	2013-03-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M113.465609
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Article: Platform for high-throughput antibody selection using synthetically-designed antibody libraries

Batonick, Melissa / Brian K. Kay / Dawn Alderman / Erika G. Holland / Margaret M. Kiss / Michael P. Weiner / Valeria Busygina

New biotechnology. 2016 Sept. 25, v. 33, no. 5

2016

Abstract	Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>1020), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 1010. To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.
Keywords	antibodies ; DNA libraries ; early selection ; Escherichia coli ; humans
Language	English
Dates of publication	2016-0925
Size	p. 565-573.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	2400836-9
ISSN	1876-4347 ; 1871-6784
ISSN (online)	1876-4347
ISSN	1871-6784
DOI	10.1016/j.nbt.2015.11.005
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Article ; Online: (with research data) Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks.

Liu, Yan / Gaines, William A / Callender, Tracy / Busygina, Valeria / Oke, Ashwini / Sung, Patrick / Fung, Jennifer C / Hollingsworth, Nancy M

PLoS genetics

2014 Volume 10, Issue 1, Page(s) e1004005

Abstract: Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a ... ...

Abstract	Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.
MeSH term(s)	Cell Cycle Proteins/genetics ; Chromatids/genetics ; Chromosome Segregation/genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; DNA-Binding Proteins/genetics ; Gene Expression Regulation, Fungal ; Homologous Recombination/genetics ; Meiosis/genetics ; Mutation ; Rad51 Recombinase/genetics ; Rad51 Recombinase/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics ; Spores/growth & development
Chemical Substances	Cell Cycle Proteins ; DMC1 protein, S cerevisiae ; DNA-Binding Proteins ; Saccharomyces cerevisiae Proteins ; Rad51 Recombinase (EC 2.7.7.-)
Language	English
Publishing date	2014-01-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2186725-2
ISSN	1553-7404 ; 1553-7390
ISSN (online)	1553-7404
ISSN	1553-7390
DOI	10.1371/journal.pgen.1004005
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Article: Hed1 regulates Rad51-mediated recombination via a novel mechanism.

Busygina, Valeria / Sehorn, Michael G / Shi, Idina Y / Tsubouchi, Hideo / Roeder, G Shirleen / Sung, Patrick

Genes & development

2008 Volume 22, Issue 6, Page(s) 786–795

Abstract: Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 ... ...

Abstract	Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination.
MeSH term(s)	Adenosine Triphosphatases/metabolism ; Cloning, Molecular ; DNA Damage ; DNA Helicases ; DNA Repair Enzymes ; Meiosis/physiology ; Mitosis/physiology ; Rad51 Recombinase/metabolism ; Recombination, Genetic/physiology ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/growth & development ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Saccharomyces cerevisiae Proteins/physiology
Chemical Substances	Saccharomyces cerevisiae Proteins ; Rad51 Recombinase (EC 2.7.7.-) ; Adenosine Triphosphatases (EC 3.6.1.-) ; RAD54 protein, S cerevisiae (EC 3.6.1.-) ; DNA Helicases (EC 3.6.4.-) ; DNA Repair Enzymes (EC 6.5.1.-)
Language	English
Publishing date	2008-03-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	806684-x
ISSN	1549-5477 ; 0890-9369
ISSN (online)	1549-5477
ISSN	0890-9369
DOI	10.1101/gad.1638708
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Zs.A 2292: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 54: Show issues

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Article ; Online: Novel attributes of Hed1 affect dynamics and activity of the Rad51 presynaptic filament during meiotic recombination.

Busygina, Valeria / Saro, Dorina / Williams, Gareth / Leung, Wing-Kit / Say, Amanda F / Sehorn, Michael G / Sung, Patrick / Tsubouchi, Hideo

The Journal of biological chemistry

2011 Volume 287, Issue 2, Page(s) 1566–1575

Abstract: During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, ...

Abstract	During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control.
MeSH term(s)	Chromatids/genetics ; Chromatids/metabolism ; Chromosomes, Fungal/genetics ; Chromosomes, Fungal/metabolism ; DNA Helicases ; DNA Repair Enzymes ; DNA, Fungal/genetics ; DNA, Fungal/metabolism ; DNA, Single-Stranded/genetics ; DNA, Single-Stranded/metabolism ; Meiosis/physiology ; Mutation ; Rad51 Recombinase/genetics ; Rad51 Recombinase/metabolism ; Recombination, Genetic/physiology ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism
Chemical Substances	DNA, Fungal ; DNA, Single-Stranded ; Saccharomyces cerevisiae Proteins ; RAD51 protein, S cerevisiae (EC 2.7.7.-) ; Rad51 Recombinase (EC 2.7.7.-) ; RAD54 protein, S cerevisiae (EC 3.6.1.-) ; DNA Helicases (EC 3.6.4.-) ; DNA Repair Enzymes (EC 6.5.1.-)
Language	English
Publishing date	2011-11-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M111.297309
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Article: Holliday junction processing activity of the BLM-Topo IIIalpha-BLAP75 complex.

Bussen, Wendy / Raynard, Steven / Busygina, Valeria / Singh, Akhilesh K / Sung, Patrick

The Journal of biological chemistry

2007 Volume 282, Issue 43, Page(s) 31484–31492

Abstract: BLM, the protein mutated in Bloom's syndrome, possesses a helicase activity that can dissociate DNA structures, including the Holliday junction, expected to arise during homologous recombination. BLM is stably associated with topoisomerase IIIalpha (Topo ...

Abstract	BLM, the protein mutated in Bloom's syndrome, possesses a helicase activity that can dissociate DNA structures, including the Holliday junction, expected to arise during homologous recombination. BLM is stably associated with topoisomerase IIIalpha (Topo IIIalpha) and the BLAP75 protein. The BLM-Topo IIIalpha-BLAP75 (BTB) complex can efficiently resolve a DNA substrate that harbors two Holliday junctions (the double Holliday junction) in a non-crossover manner. Here we show that the Holliday junction unwinding activity of BLM is greatly enhanced as a result of its association with Topo IIIalpha and BLAP75. Enhancement of this BLM activity requires both Topo IIIalpha and BLAP75. Importantly, Topo IIIalpha cannot be substituted by Escherichia coli Top3, and the Holliday junction unwinding activity of BLM-related helicases WRN and RecQ is likewise impervious to Topo IIIalpha and BLAP75. However, the topoisomerase activity of Topo IIIalpha is dispensable for the enhancement of the DNA unwinding reaction. We have also ascertained the requirement for the BLM ATPase activity in double Holliday junction dissolution and DNA unwinding by constructing, purifying, and characterizing specific mutant variants that lack this activity. These results provide valuable information concerning how the functional integrity of the BTB complex is governed by specific protein-protein interactions among the components of this complex and the enzymatic activities of BLM and Topo IIIalpha.
MeSH term(s)	Adenosine Triphosphatases/chemistry ; Adenosine Triphosphatases/genetics ; Adenosine Triphosphatases/isolation & purification ; Adenosine Triphosphatases/metabolism ; Adenosine Triphosphate/metabolism ; Bloom Syndrome/genetics ; Bloom Syndrome/metabolism ; Carrier Proteins/metabolism ; DNA Helicases/chemistry ; DNA Helicases/genetics ; DNA Helicases/isolation & purification ; DNA Helicases/metabolism ; DNA Topoisomerases, Type I/genetics ; DNA Topoisomerases, Type I/physiology ; DNA, Cruciform/metabolism ; DNA-Binding Proteins ; Escherichia coli/metabolism ; Genetic Variation ; Histidine/chemistry ; Humans ; Hydrolysis ; Mutation ; Nuclear Proteins/metabolism ; Protein Binding ; RecQ Helicases ; Recombination, Genetic ; Substrate Specificity
Chemical Substances	Carrier Proteins ; DNA, Cruciform ; DNA-Binding Proteins ; Nuclear Proteins ; RMI1 protein, human ; Histidine (4QD397987E) ; Adenosine Triphosphate (8L70Q75FXE) ; Adenosine Triphosphatases (EC 3.6.1.-) ; Bloom syndrome protein (EC 3.6.1.-) ; DNA Helicases (EC 3.6.4.-) ; RecQ Helicases (EC 3.6.4.12) ; DNA Topoisomerases, Type I (EC 5.99.1.2)
Language	English
Publishing date	2007-08-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M706116200
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Article ; Online: Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1.

Busygina, Valeria / Gaines, William A / Xu, Yuanyuan / Kwon, Youngho / Williams, Gareth J / Lin, Sheng-Wei / Chang, Hao-Yen / Chi, Peter / Wang, Hong-Wei / Sung, Patrick

DNA repair

2013 Volume 12, Issue 9, Page(s) 707–712

Abstract: The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that ...

Abstract	The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.
MeSH term(s)	Adenosine Triphosphate/chemistry ; Calcium/chemistry ; Cell Cycle Proteins/chemistry ; Cell Cycle Proteins/isolation & purification ; Cell Cycle Proteins/physiology ; Chromatography, Gel ; DNA Helicases/chemistry ; DNA Repair Enzymes/chemistry ; DNA Topoisomerases/chemistry ; DNA, Fungal/chemistry ; DNA, Fungal/ultrastructure ; DNA, Single-Stranded/chemistry ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/isolation & purification ; DNA-Binding Proteins/physiology ; Homologous Recombination ; Humans ; Hydrolysis ; Protein Binding ; Saccharomyces cerevisiae/enzymology ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/isolation & purification ; Saccharomyces cerevisiae Proteins/physiology
Chemical Substances	Cell Cycle Proteins ; DMC1 protein, S cerevisiae ; DNA, Fungal ; DNA, Single-Stranded ; DNA-Binding Proteins ; Saccharomyces cerevisiae Proteins ; Adenosine Triphosphate (8L70Q75FXE) ; RAD54 protein, S cerevisiae (EC 3.6.1.-) ; DNA Helicases (EC 3.6.4.-) ; DNA Topoisomerases (EC 5.99.1.-) ; RDH54 protein, S cerevisiae (EC 5.99.1.-) ; DNA Repair Enzymes (EC 6.5.1.-) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2013-06-12
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2071608-4
ISSN	1568-7856 ; 1568-7864
ISSN (online)	1568-7856
ISSN	1568-7864
DOI	10.1016/j.dnarep.2013.05.004
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Zs.A 5555: Show issues

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Article ; Online: AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

Holland, Erika G / Buhr, Diane L / Acca, Felicity E / Alderman, Dawn / Bovat, Kristin / Busygina, Valeria / Kay, Brian K / Weiner, Michael P / Kiss, Margaret M

Journal of immunological methods

2013 Volume 394, Issue 1-2, Page(s) 55–61

Abstract: Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too ... ...

Abstract	Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.
MeSH term(s)	Escherichia coli/genetics ; Mutagenesis ; Peptide Library ; Polymerase Chain Reaction ; Recombinant Proteins/biosynthesis
Chemical Substances	Peptide Library ; Recombinant Proteins
Language	English
Publishing date	2013-08-30
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	120142-6
ISSN	1872-7905 ; 0022-1759
ISSN (online)	1872-7905
ISSN	0022-1759
DOI	10.1016/j.jim.2013.05.003
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