Concise Overview of Methodologies Employed in the Study of Bacterial DNA Replication

Abstract

DNA replication is a fundamental process in the cell on which the functioning of the entire cell as well as the maintenance of the entire species depends. This process is synchronized with all other processes within the cell as well as with external, environmental factors. This complex network of interconnections presents significant challenges in the field of DNA replication research, both in terms of identifying an appropriate approach to a question posed and in terms of methodology. This article aims to provide a roadmap to assist in navigating (to help overcome) these challenges and in selecting an appropriate research methodology. It should help to establish a research pathway, starting with arranging the host genetic background for analysis at different cellular levels, which can be achieved using complex or simple single-purpose techniques.

Keywords: DNA replication; bacteria; electrophoresis; flow cytometry; genetic engineering; methods; microscopy; techniques.

Figure 1

Cell cycle and DNA replication: slow (A) and fast-growing (B) bacterial cells. Panel (C) depicts an origin-to-terminus ratio in slow and fast-growing bacteria. B phase-blue colour, C phase green colour, D phase-orange colour.

Figure 2

Steps in the assembly of the replication complex in Escherichia coli, covering events from the recognition of the ori sequence by the initiator protein DnaA, DnaB helicase assembly, the local unzipping of the double-stranded DNA structure (light purple DNA strands) to the primer synthesis by the DnaG primase, and the early elongation of both strands on the DNA template. PolIII HE- —DNA polymerase III holoenzyme; NrdA, NrdB-subunits of ribonucleoside diphosphate reductase 1; DiaA, had -replication initiation regulatory proteins. DiaA. stimulates DnaA assembly on oriC, whereas Hda inhibits the reinitiation of DNA replication by promoting DnaA-ATP hydrolysis in the process termed RIDA. Thick, black-shaded arrows indicate the transition to the subsequent stage in the initiation of DNA replication. Thin black arrows indicate the attachment or detachment of additional proteins/complexes during the subsequent stages in the formation of the replication complex. See text for more details.

Figure 3

Distribution of ter sites in the Escherichia coli chromosome. Light purple indicates single-stranded DNA fragments in locally separated (unwound) DNA double helix at the ori site. The dark turquoise line of the circle marks the DNA helix of the chromosome.

Figure 4

(A) Type IIS RE cutting scheme. (B) The Golden Gate cloning scheme. The successive stages of the experiment are indicated by numbers. First, vectors and insert(s) are digested with type IIS RE of choice, which gives the complementary sequence overhangs (step 1 indicated with gray arrow) that can be ligated using T4 DNA ligase to a cloned vector (step 2 indicated with gray arrow). Since type IIS RE is cut outside the recognition sequence, the specific overhangs can be freely designed, which gives the possibility to assemble several fragments in a one-pot reaction. DNA fragments of the vector and insert complementary to each other were marked with identical colours. Ligation occurs between fragments of the same colours. The shorter fragment of the vector DNA located between the complementary sequences highlighted in colour, is cut out during restriction digestion.

Figure 5

Restriction-free (RF) cloning scheme. The successive stages of the experiment are indicated by numbers. The first two stages, numbered 1 and 2, represent two consecutive PCR reactions. The third stage, numbered 3, involves the selection of the product by digestion with the DpnI enzyme. The fourth stage, numbered 4, entails the transformation of Escherichia coli cells with the obtained construct. Overhang-containing primers are indicated by thin arrows. Thick arrows indicate the transition between subsequent cloning steps, describes as numbers above.

Figure 6

Groups of methods taken into consideration in this paper—see details in the text.

Figure 7

Electrophoretic-based methods considered in this paper. A brief characterization of each method is included. See text for details.