Timothy Lohman - Academia.edu (original) (raw)
Papers by Timothy Lohman
Biophysical Journal, Feb 1, 2018
Proceedings of the National Academy of Sciences of the United States of America, Sep 17, 1996
Seminars in Cell & Developmental Biology, Feb 1, 2019
Nature Communications, Aug 13, 2013
Nucleic Acids Research, Jul 22, 2019
Nature Communications, Dec 1, 2021
Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to in... more Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Ku has been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rate...
bioRxiv (Cold Spring Harbor Laboratory), Oct 16, 2023
Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structur... more Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB ΔNuc CD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant (RecB D1080A CD), which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤ 60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecB ΔNuc CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB ΔNuc CD also unwinds DNA ~two-fold slower than RecBCD on long DNA (~20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB D1080A CD unwinding are intermediate between RecBCD and RecB ΔNuc CD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecB Δ Nuc CD may mimic a post-chi state of RecBCD.
Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that i... more Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5’ to 3’ translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and ...
ABSTRACTThe heterodimeric actin capping protein (CP) is regulated by a set of proteins that conta... more ABSTRACTThe heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not...
Proceedings of the National Academy of Sciences, 2019
In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex ... more In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. It must undergo a repair and reassembly process following photodamage, many facets of which remain unknown. We have discovered a PSII subcomplex that lacks 5 key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment–protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low molecular mass subunits, and the assembly factor Psb27. Immunoblotting, mass spectrometry, and ultrafast spectroscopic results support the absence of a functional reaction center in this complex, which we call the “no reaction center” complex (NRC). Analytical ultracentrifugation and clear native PAGE analysis show that NRC is a stable pigment–protein complex and not a mixture of free CP47 and CP43 proteins. NRC appears ...
Biophysical Journal, 2019
Biophysical Journal, 2017
Biophysical Journal, 2016
Biophysical Journal, 2014
in a sequence specific manner. Several methods of varying complexity are available to engineer ZF... more in a sequence specific manner. Several methods of varying complexity are available to engineer ZFPs that can target all the 64 codons in the genome. Although ZFPs are becoming a powerful tool for site specific modification in the genome, several challenges remain before the full potential of ZFPs can be realized. The engineered ZFPs generated using the present design platforms target mostly base triplets with 5' Guanine (GNN, where N is any nucleotide) and the non-GNN or AT rich modules are difficult to target. In the present project we attempt to address this challenge by designing linker regions between the ZFP motifs to target non-contiguous base pairs in the DNA. This will increase the number of targetable DNA sequences by an order of magnitude and will help to realize the full potential of ZFPs. Using structure based methods, we provide an extensive library of possible linker molecules that can be introduced between the individual zinc finger motifs to skip up to 10 base pairs between adjacent zinc finger protein recognition sites in the DNA sequences. We also performed a proof of principle experiment to validate the binding affinity and specificity of one of the computationally designed ZFP to its target DNA sequence.
Biophysical Journal, 2019
Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ... more Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB) 35) mode favored at low [NaCl] and high SSB/DNA ratios displays high ''unlimited'' nearest-neighbor cooperativity (u 35), forming long protein clusters, whereas the 65-site-size ((SSB) 65) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays ''limited'' cooperativity (u 65), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB) 65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearestneighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT) 70 and (dT) 140. We find that the (SSB) 35 mode always shows extremely high ''unlimited'' cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-DL, bind in the (SSB) 65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (u 65/35), with u 65 << u 65/35 < u 35. The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearestneighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity.
Nucleic Acids Research, 2019
Escherichia coli single strand (ss) DNA binding (SSB) protein protects ssDNA intermediates and re... more Escherichia coli single strand (ss) DNA binding (SSB) protein protects ssDNA intermediates and recruits at least 17 SSB interacting proteins (SIPs) during genome maintenance. The SSB C-termini contain a 9 residue acidic tip and a 56 residue intrinsically disordered linker (IDL). The acidic tip interacts with SIPs; however a recent proposal suggests that the IDL may also interact with SIPs. Here we examine the binding to four SIPs (RecO, PriC, PriA and χ subunit of DNA polymerase III) of three peptides containing the acidic tip and varying amounts of the IDL. Independent of IDL length, we find no differences in peptide binding to each individual SIP indicating that binding is due solely to the acidic tip. However, the tip shows specificity, with affinity decreasing in the order: RecO > PriA ∼ χ > PriC. Yet, RecO binding to the SSB tetramer and an SSB–ssDNA complex show significant thermodynamic differences compared to the peptides alone, suggesting that RecO interacts with anot...
Nucleic Acids Research
Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA main... more Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO–ssDNA aggregates occur that form with higher propensity o...
Nucleic Acids Research, 2020
Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recrui... more Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wi...
Biophysical Journal, Feb 1, 2018
Proceedings of the National Academy of Sciences of the United States of America, Sep 17, 1996
Seminars in Cell & Developmental Biology, Feb 1, 2019
Nature Communications, Aug 13, 2013
Nucleic Acids Research, Jul 22, 2019
Nature Communications, Dec 1, 2021
Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to in... more Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Ku has been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rate...
bioRxiv (Cold Spring Harbor Laboratory), Oct 16, 2023
Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structur... more Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecB ΔNuc CD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant (RecB D1080A CD), which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤ 60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecB ΔNuc CD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecB ΔNuc CD also unwinds DNA ~two-fold slower than RecBCD on long DNA (~20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecB D1080A CD unwinding are intermediate between RecBCD and RecB ΔNuc CD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecB Δ Nuc CD may mimic a post-chi state of RecBCD.
Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that i... more Replication protein A (RPA) is a eukaryotic single stranded (ss) DNA binding (SSB) protein that is essential for all aspects of genome maintenance. RPA binds ssDNA with high affinity but can also diffuse along ssDNA. By itself, RPA is capable of transiently disrupting short regions of duplex DNA by diffusing from a ssDNA that flanks the duplex DNA. Using single molecule total internal reflection fluorescence and optical trapping combined with fluorescence approaches we show that S. cerevisiae Pif1 can use its ATP-dependent 5’ to 3’ translocase activity to chemo-mechanically push a single human RPA (hRPA) directionally along ssDNA at rates comparable to those of Pif1 translocation alone. We further show that using its translocation activity Pif1 can push hRPA from a ssDNA loading site into a duplex DNA causing stable disruption of at least 9 bp of duplex DNA. These results highlight the dynamic nature of hRPA enabling it to be readily reorganized even when bound tightly to ssDNA and ...
ABSTRACTThe heterodimeric actin capping protein (CP) is regulated by a set of proteins that conta... more ABSTRACTThe heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not...
Proceedings of the National Academy of Sciences, 2019
In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex ... more In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. It must undergo a repair and reassembly process following photodamage, many facets of which remain unknown. We have discovered a PSII subcomplex that lacks 5 key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment–protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low molecular mass subunits, and the assembly factor Psb27. Immunoblotting, mass spectrometry, and ultrafast spectroscopic results support the absence of a functional reaction center in this complex, which we call the “no reaction center” complex (NRC). Analytical ultracentrifugation and clear native PAGE analysis show that NRC is a stable pigment–protein complex and not a mixture of free CP47 and CP43 proteins. NRC appears ...
Biophysical Journal, 2019
Biophysical Journal, 2017
Biophysical Journal, 2016
Biophysical Journal, 2014
in a sequence specific manner. Several methods of varying complexity are available to engineer ZF... more in a sequence specific manner. Several methods of varying complexity are available to engineer ZFPs that can target all the 64 codons in the genome. Although ZFPs are becoming a powerful tool for site specific modification in the genome, several challenges remain before the full potential of ZFPs can be realized. The engineered ZFPs generated using the present design platforms target mostly base triplets with 5' Guanine (GNN, where N is any nucleotide) and the non-GNN or AT rich modules are difficult to target. In the present project we attempt to address this challenge by designing linker regions between the ZFP motifs to target non-contiguous base pairs in the DNA. This will increase the number of targetable DNA sequences by an order of magnitude and will help to realize the full potential of ZFPs. Using structure based methods, we provide an extensive library of possible linker molecules that can be introduced between the individual zinc finger motifs to skip up to 10 base pairs between adjacent zinc finger protein recognition sites in the DNA sequences. We also performed a proof of principle experiment to validate the binding affinity and specificity of one of the computationally designed ZFP to its target DNA sequence.
Biophysical Journal, 2019
Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ... more Escherichia coli single-strand (ss) DNA-binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes differing in occluded site size and cooperativity. The 35-site-size ((SSB) 35) mode favored at low [NaCl] and high SSB/DNA ratios displays high ''unlimited'' nearest-neighbor cooperativity (u 35), forming long protein clusters, whereas the 65-site-size ((SSB) 65) mode in which ssDNA wraps completely around the tetramer is favored at higher [NaCl] (>200 mM) and displays ''limited'' cooperativity (u 65), forming only dimers of tetramers. In addition, a non-nearest-neighbor high cooperativity can also occur in the (SSB) 65 mode on long ssDNA even at physiological salt concentrations in the presence of glutamate and requires its intrinsically disordered C-terminal linker (IDL) region. However, whether cooperativity exists between the different modes and the role of the IDL in nearestneighbor cooperativity has not been probed. Here, we combine sedimentation velocity and fluorescence titration studies to examine nearest-neighbor cooperativity in each binding mode and between binding modes using (dT) 70 and (dT) 140. We find that the (SSB) 35 mode always shows extremely high ''unlimited'' cooperativity that requires the IDL. At high salt, wild-type SSB and a variant without the IDL, SSB-DL, bind in the (SSB) 65 mode but show little cooperativity, although cooperativity increases at lower [NaCl] for wild-type SSB. We also find significant intermode nearest-neighbor cooperativity (u 65/35), with u 65 << u 65/35 < u 35. The intrinsically disordered region of SSB is required for all cooperative interactions; however, in contrast to the non-nearest-neighbor cooperativity observed on longer ssDNA, glutamate does not enhance these nearestneighbor cooperativities. Therefore, we show that SSB possesses four types of cooperative interactions, with clear differences in the forces stabilizing nearest-neighbor versus non-nearest-neighbor cooperativity.
Nucleic Acids Research, 2019
Escherichia coli single strand (ss) DNA binding (SSB) protein protects ssDNA intermediates and re... more Escherichia coli single strand (ss) DNA binding (SSB) protein protects ssDNA intermediates and recruits at least 17 SSB interacting proteins (SIPs) during genome maintenance. The SSB C-termini contain a 9 residue acidic tip and a 56 residue intrinsically disordered linker (IDL). The acidic tip interacts with SIPs; however a recent proposal suggests that the IDL may also interact with SIPs. Here we examine the binding to four SIPs (RecO, PriC, PriA and χ subunit of DNA polymerase III) of three peptides containing the acidic tip and varying amounts of the IDL. Independent of IDL length, we find no differences in peptide binding to each individual SIP indicating that binding is due solely to the acidic tip. However, the tip shows specificity, with affinity decreasing in the order: RecO > PriA ∼ χ > PriC. Yet, RecO binding to the SSB tetramer and an SSB–ssDNA complex show significant thermodynamic differences compared to the peptides alone, suggesting that RecO interacts with anot...
Nucleic Acids Research
Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA main... more Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO–ssDNA aggregates occur that form with higher propensity o...
Nucleic Acids Research, 2020
Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recrui... more Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wi...