Question

Using model materials to demonstrate DNA replication

1.    Present a detailed analysis of DNA replication at one replication fork. Use drawing, descriptions, and/or captions detailing the process.

2.    In the analysis include the following:

a.    Show how the leading and lagging strands are synthesized

b.    Show the proteins (enzymes) involved in DNA replication and what their functions are

Answer 1

Transmission of chromosomal DNA from one generation to next generation is very crucial for the cell propagation. This can only be achieved when chromosomal DNA is accurately replicated, providing two copies f the entire genome for faithful distribution into each daughter cell.

In bacterial cell the circular chromosomes contains a unique origin to the terminus. Therefore, the whole bacterial genome has a single replicon means monorepliconic.

The genome of E. coli is replicated bidirectionally by using a single oriC, this type of replication in known as theta replication.

oriC contains11 5’-GATC-3’ repeats that are methylated on adenine on both strands. Only fully methylated origin can initiates the replication.

The origin of replication is a cis acting sequences. In E. coli single origin of replication present in the chromosome is referred to as oriC. The E. coli oriC contains two short repeat motifs, one is made up of 9 nucleotides and another is of 13 nucleotides. The 9 nucleotides repeat is the binding site for the DnaA protein. It is an initiator protein which initiates replication in E. coli at oriC. As the binding of DnaA at 9-mers sequences of double helix opens up within the tandem array of three AT-rich, 13 nucleotides repeats located at one end of the oriC sequences. This process requires ATP and forms open complex. After the melting of the two strand of the E. coli chromosomes to generate unpaired template strands is mediated by the DnaB protein which is a helicase. One molecule of DnaB, clamps around each of the two single strands in the open complex formed between DnaA and oriC which requires ATP and DnaC (helicase loader). Helicase move around the DNA strand utilizing the energy of ATP hydrolysis to separate the strands. The separated strands are inhibited from subsequently reannealig by single strand binding protein, which binds with both separated strands. DNA polymerase can only elongate existing primer strand of DNA or RNA. The primer used during DNA replication in both prokaryotic and eukaryotic are short RNA molecules, which are synthesized by RNA polymerase called as primase.

Proteins and their function

Initiator proteins- DnaA, single strand binding protein (SSBs), DnaB (helicase), DnaC and DnaG (primase) (functions of all these are mentioned in above paragraph)

Proteins required for chain elongation and connecting Okazaki fragments are DNA polymerase, single strand binding protein (SSBs), DNA gyrase (a swivel to relieve positive super coiling) and DNA ligase (ligates the okazaki fragments)

Proteins required for the termination of replication and segregation of daughter molecules are terminus binding protein (tus) and DNA Topoisomerase IV.