Test questions

1. What are characteristic features of protein metabolism?

2. Define the concept of "nitrogen balance".

3. What are the main reasons for the breadown of tissue proteins?

4. Describe the process of protein digestion in the gastrointestinal tract.

5. What is the mechanism of activation of the gastrointestinal tract proteases?

6. What conversions amino acids undergo under the influence of intestinal microflora?

7. What proteins are the most adequate for a human?

8. Enumerate types of amino acid deamination.

9. What is the significance of transamination reactions?

10. Give examples of reactions involving carboxyl group of amino acids.

11. In what form ammonia and amine nitrogen come from peripheral tissues to liver for urea synthesis?

12. Why is the content of urea cycle enzymes increases either at abundant protein diet, or at starvation?

13. What substances are formed during the degradation of hemoglobin?

14. What enzymes are involved in the degradation of nucleic acids?

15. What are end products of degradation of purine and pyrimidine bases?

16. What compounds are donor of atoms of pyrimidine and purine bases rings?

3. Template biosynthesis

3.1. Biosynthesis of nucleic acids

The biosynthesis of nucleic acids takes place only in the presence of all four species of deoxyribonucleoside triphosphates (DNA synthesis) or ribonucleoside triphosphates (RNA synthesis). Enzymes DNA or RNA polymerases are involved in the biosynthesis. The presence of polynucleotide which plays the role of the template is necessary. This ensures the nucleic acids biosynthesis with a strictly defined sequence of nucleotide residues in the molecule.

3.1.1. Dna biosynthesis (replication)

The general scheme of DNAbiosynthesis (A. Kornberg, 1958): DNA double helix unwinds, the strands go apart. The single DNA strands serve as template for the synthesis of new chains. The result is two double-stranded DNA molecules that are identical to the original molecule. The nucleotide sequence of new chains is determined by the rule of base complementary and the sequence of nucleotides of existing chain. Synthesis of DNA is called replication.

Homological replication is an endless repeating process of doubling the number of molecules by direct copying their structure.

Kornberg A. and S. Ochoa were awarded the Nobel Prize in 1959 for outstanding contribution to solving the problems of the biosynthesis of DNA and RNA.

Enzymes of DNA biosynthesis. Prokaryotes.

Helicase separates DNA strands at the replication fork.

RNA polymerase (primase) catalyzes the synthesis of oligoribonucleotides (10 to 60 nucleotides), i.e., the primer from which DNA synthesis begins.

Primasome is the complex which includes about 20 polypeptides. It is involved in the formation of DNA specific secondary structure, suitable for recognition by primase.

DNA polymerase Icatalyzes the cleavage of the primer, removing wrongly connected nucleotide residues and filling in gaps (DNA polymerase activity).

DNA polymerase II removes wrongly placed nucleotide bases and edits mistakes, i.e., carries out DNA repair.

DNA polymerase III catalyzes the synthesis of the leading and lagging DNA strands at replication.

DNA ligase joins the two DNA strands, or closes the two ends of one chain at replication or repair.

Topoisomerases cut or reseal DNA strands and create or destroy supercoiling.

Eukaryotic DNA polymerases: α, β, γ, ε, .

In DNA replication two main types of polymerases are involved: αand . DNA polymerase catalyzes the synthesis of DNA leading strand, and DNA polymerase α catalyzes the synthesis of DNA lagging strand, being the part of primasome. None of the eukaryotes DNA polymerases, unlike prokaryotes, has nuclease activity.

DNA polymerase γ replicates mitochondrial DNA.

DNA polymerase ε replaces DNA polymerase  in some cases and performs proof-reading function.

DNA ligase fills the gaps in one strand of DNA and closes linear DNA molecule into a ring structure.

Protein factors required for DNA biosynthesis.

Single stranded DNA binding (SSB) protein weakens the interaction of DNA strands and activates the DNA polymerases II and III.

DNA-unwinding protein has nuclease activity. It breaks the linkage in one DNA strand that provides the unwinding of the molecule.

DNA-winding protein provides DNA supercoiling.

More than 40 enzymes and protein factors, combined in a replisome involved in DNA replication in total.

The steps of DNA biosynthesis

Initiation. The two complementary DNA strands separate at the site of replication. SSB protein, DNA unwinding protein, DNA polymerase complex, primase and primosome attach to the single-stranded DNA fragment. Replicative fork is formed (Fig. 2).

A primer (a short RNA fragment) is synthesized at the parent DNA strand with the participation of primase (RNA polymerase). Then, the new strand is synthesized on the parent DNA strand by means of DNA polymerase III.

Elongation. The polymerization process goes only in the 5'  3' direction. Both chains are replicated simultaneously. Their synthesis is in the opposite directions. Synthesis of the leading strand of DNA is carried out continuously.

Fig. 2. Replicative fork structure

Lagging strand is formed in a direction opposite to the movement of the replication fork. Synthesis is fragmentary. These fragments are called Okazaki pieces in honor of the Japanese biochemist, who first proposed the scheme of the biosynthesis of DNA, which has overcome difficulties in synthesis of antiparallel strands of DNA molecule. Length of Okazaki pieces is 150-200 thousand nucleotides in eukaryotes and 1000-2000 in bacteria.

Elongation ends with separation of primers and filling the vacant places by complementary deoxyribonucleotides under the action of DNA polymerase I. DNA fragments are combined using DNA ligase.

The accuracy of DNA replication is an error on the 10¹⁰ reactions. The admitted error can be corrected in the repair processes.

Replication begins at sites of DNA having special nucleotide sequence, and called origin. Origins are located approximately from 100 000 nm. Part of DNA between the neighboring origins is called replicon. Each replicon is replicated by two replicative complexes moving toward each other. A replicon is replicated in 2 hours and the same amount of time is required for the replication of DNA molecules of any length. In fact, replication of the human genome in vivo lasts 6-8 hours. If the DNA molecule is replicated by one replicative complex, then it would take 10 days.

Termination. Termination of DNA replication is programmed by specific nucleotide sequence.