DNA Replication - Biochemistry

Prokaryotic DNA replication occurs in the cytoplasm, since these cells lack nuclei. Prokaryotic genomes are comprised of a single circular chromosome, with one origin of replication. Translation is the process of protein synthesis, which occurs on ribosomes free in the cytosol (or on ribosomes embedded in the rough endoplasmic reticulum in eukaryotes).

The only true statement is that prokaryotic DNA replication is faster than eukaryotic DNA replication.

Primase is actually an RNA polymerase, not a DNA polymerase. Primase creates an RNA primer which is used to replicate short-stranded DNA. Primers serve as the starting point for DNA synthesis, so RNA polymerases wouldn’t require them. There are a number of means by which DNA is repaired including direct repair, excision repair, and homologous recombination. DNA repair, however, does not involve the use of RNA polymerases. All rRNA (except 5S rRNA) is synthesized by RNA polymerase I (or to be specific, the polymerase creates a pre-RNA which matures into rRNA), while the precursors of mRNA are synthesized by RNA polymerase II. Primers are short, complementary RNA sequences that serve as the starting point for DNA synthesis; the DNA polymerase begins replication at the primer’s 3’ end, and uses the opposite strand as a template. Without the primer, the DNA polymerase would not have an existing strand of nucleotides onto which it could attach new nucleotides.

To answer this question, it's important to understand that DNA replicates in a semi-conservative fashion. This means that the two complementary strands of DNA split apart, and a new complementary strand is added to each of the parent strands. Thus, each daughter DNA molecule will be composed of one parent strand, and one newly synthesized strand. Since we know that adenine base pairs with thymine, and guanine base pairs with cytosine, the ratio of is expected to remain the same, provided no mutations occur.

DNA replication is the process of producing a duplicate copy of a DNA strand. DNA double helix is first unwound by breaking the hydrogen bonds between nitrogenous bases, giving two parent strands. Next, these unwound DNA strands are utilized as a template strand (parent strand) to create a daughter strand that is identical to the parent strand. After completion of the replication, the parent strand and daughter strand hybridize (hydrogen bonds re-form between bases) and form a double helix. Note that the original parent strands never re-hybridize.

Epigenetic changes refer to alterations in DNA molecules or histones. These alterations can enhance or suppress transcription of DNA to RNA. DNA replication is unaffected by epigenetic changes.

As mentioned, each parent strand produces an identical, daughter strand that ultimately re-hybridizes with the parent strand (forms double helix structure); therefore, each parent strand only produces one daughter strand.

Unwinding of the double helix involves breaking the hydrogen bonds between nitrogenous bases from adjacent DNA molecules. Recall that hydrogen bonds occur between a hydrogen atom and either a nitrogen, oxygen, or fluorine atom. The nitrogenous bases found in DNA and RNA molecules do not contain any fluorine atoms; therefore, fluorine (although it is involved in hydrogen bonds in other molecules) is not involved in hydrogen bonding between nitrogenous bases.

Cell cycle has four main phases: G1, S, G2 phases, and mitosis. DNA replication occurs during the S phase of the cell cycle, which occurs between G1 and G2 phase; therefore, DNA replication occurs between G1 and G2 phases.

Note that there is a cell checkpoint before the beginning of S phase (at the end of G1 phase) to ensure that the DNA molecules in the cell’s nucleus are prepared and stable for DNA replication. If it fails this checkpoint, the cell stays in the G1 phase until the DNA is ready for replication.

DNA bases are joined together by hydrogen bonds. Adenine and thymine are bound by two hydrogen bonds, and cytosine and guanine are bound by three hydrogen bonds. These bases make up the "rungs" of the twisted ladder that is DNA. Therefore, if the bases need to be separated, hydrogen bonds must be broken to separate the two strands. Also, remember that these bases need to be rejoined, so the bonds between them need not be as strong as covalent bonds since they are continuously broken and reformed.

Hydrogen bonds are found between two strands of DNA (between nitrogenous bases). Peptide bonds and disulfide bonds are found in proteins. Ionic bonds are not found in between DNA strands, and van der Waals interactions are too weak to hold two DNA strands together.

Primase acts to initialize DNA synthesis. DNA synthesis requires a free 3' hydroxyl group bound to the template strand to begin. This is accomplished by having a temporary strand of RNA called the primer attached to the template DNA - it is created by primase. It must be the 3' hydroxyl end that is free because DNA synthesis occurs in the 5' to 3' direction, and that 3' hydroxyl group is the substrate for DNA polymerase.

A double helix DNA structure can be coiled or even supercoiled. In order to relieve the tension that is inevitably formed by this coiling, topoisomerase acts upon the DNA to relieve the stress that has been created. Helicase unwinds DNA before replication. DNA polymerase elongates the DNA during replication. RNA polymerase makes RNA from a template strand of DNA. Primase creates a temporary primer to begin DNA synthesis/replication.

In DNA replication, the role of helicase is to unwind the strand by breaking the hydrogen bonds that hold that two strands together.

All of the other answer choices describe a role performed by a different DNA replication enzyme. Let's go ahead and review these.

• Lays down an RNA primer so that the synthesis of complementary daughter DNA can occur

This enzyme is called Primase.

• Stitches together the various daughter DNA fragments into a single strand

This enzyme is called DNA Ligase. It is able to join the okazaki fragments formed on the lagging strand, as well as any other areas where there is a break in the strand.

• Holds the parent DNA strands in place during replication to prevent them from associating with one another

This enzyme is known as single-strand binding protein.

DNA polymerase I is in prokaryotes only. It degrades the RNA primer and fills in the gap with DNA. DNA polymerase III has synthesis and proofreads with exonuclease. This is also in prokaryotes only. It elongates the leading strand by adding deoxynucleotides to the 3’ end. It elongates the lagging strand until it reaches primer of preceding fragment. exonuclease activity “proofreads” each added nucleotide. The function of DNA polymerase II is unknown.

Remember, it’s complementary and antiparallel. Therefore, when writing the complement of the DNA sequence, it’s 3’ to 5’, so you must change answer to be 5’ to 3’.

Because the lagging strand is created in various separate segments during DNA replication, after polymerization they must be joined together by an enzyme. The enzyme that is responsible for these connections is DNA ligase.

Topoisomerase I functions as a stress reliever during DNA replication. DNA coils and supercoils, and without topoisomerase, the tension caused by this winding would not be able to dissipate.

The correct matches between prokaryotic DNA polymerase type and function are:

DNA polymerase I - fills in gaps in lagging strand

DNA polymerase II - DNA repair

DNA polymerase III - primary enzyme for DNA synthesis

Note: The functions of certain DNA polymerases in eukaryotes and prokaryotes are not the same.

Ribonucleotide reductase regulates the rate of DNA synthesis and the total DNA to cell mass ratio. The enzyme converts adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), uridine diphosphate (UDP). The ribonucleotide thymidine diphosphate is not a substrate for this enzyme. Thymidine nucleotides are products of another enzyme: thymidylate kinase.

Purines can be synthesized de novo from ribose phosphate. 5-phosphoribosylamine is converted to inosine monophosphate, which is an intermediary for adenine monophosphate and guanine monophosphate production. The reaction requires the presence of glycine, aspartate and glutamine, but not lysine.

Phosphoribosyl pyrophosphate (PRPP) is a precursor of both purines (adenine and guanine), as well as pyrimidines (cytosine, uracil, and thymine) in nucleotide synthesis. In certain enzyme deficiencies, levels of PRPP can increase leading indirectly to uric acid production and gout.