Related Experiment Video
Updated: Jun 10, 2025
08:14
Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
Published on: February 25, 2017
7.3K
Structural and functional insights into the interaction between the bacteriophage T4 DNA processing proteins gp32 and
Xiaoping He1, Mi-Kyung Yun2, Zhenmei Li1
1Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place MS311, Memphis, TN 38105, USA.
Nucleic Acids Research
|October 17, 2024
Summary
Bacteriophage T4 gp32 protein binds single-stranded DNA (ssDNA) and recruits the Dda helicase. Structural studies reveal how gp32 interacts with Dda, elucidating T4 DNA processing mechanisms.
Area of Science:
- Molecular Biology
- Structural Biology
- Virology
Background:
- Bacteriophage T4 is a crucial model for DNA processing research.
- The gp32 protein is essential for T4 DNA replication, repair, and recombination.
- gp32 binds single-stranded DNA (ssDNA), protecting it and facilitating protein recruitment.
Purpose of the Study:
- To elucidate the structural basis of the interaction between T4 gp32 and the Dda helicase.
- To understand how gp32 recruits and regulates Dda activity during DNA processing.
- To characterize the complete ssDNA binding surface of gp32 in complex with Dda.
Main Methods:
- Purification and crystallization of a gp32-Dda-ssDNA complex.
- Low-resolution X-ray crystallography.
- Analytical ultracentrifugation.
- Surface plasmon resonance (SPR) for peptide binding analysis.
- Dda-dependent DNA unwinding assays.
Main Results:
- A low-resolution structure revealed the C-terminal interaction between gp32 and Dda.
- SPR identified an optimal Dda-binding peptide from gp32's C-terminus.
- Crystal structures confirmed the gp32-Dda and Dda-peptide interactions.
- DNA unwinding assays demonstrated gp32's role in sequestering ssDNA generated by Dda.
- The study reveals the complete ssDNA binding surface of gp32 and insights into gp32-gp32 interactions.
Conclusions:
- The structural and functional data provide a comprehensive understanding of gp32-Dda interactions in T4 DNA processing.
- gp32 plays a dual role in protecting ssDNA and recruiting/regulating helicases.
- The findings offer insights into the assembly and regulation of viral DNA processing machinery.
Related Concept Videos
01:20Viral Replication: Lytic Cycle
7
Bacteriophages, or phages, are viruses that specifically infect bacteria. Among them, T-even bacteriophages, such as T4, exhibit a well-characterized lytic replication cycle in Escherichia coli (E. coli). This process ensures the rapid proliferation of the virus while ultimately leading to the destruction of the bacterial host.Attachment and DNA InjectionThe infection process begins with the recognition and binding of the T4 phage to the E. coli cell surface. Tail fibers of the phage...
7
02:10Translesion DNA Polymerases
9.9K
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
9.9K
02:37Restarting Stalled Replication Forks
5.8K
DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
5.8K
03:01The Replisome
33.1K
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
33.1K
01:14Rab Proteins
3.9K
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
3.9K
02:31Homologous Recombination
50.3K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
50.3K