DNA Replication Fork

Abstract

Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Multiple linear chromosomes, a large number of regulatory elements, and chromosomal packaging are challenges that the eukaryotic DNA replication fork machinery must successfully overcome. The replication machinery, the “replisome” complex, is composed of many specialized proteins with functions to support replication by DNA polymerases.

Efficient replisome progression relies on close coordination between the various replisome factors. Furthermore, replisome progression must occur in less than ideal templates at various genomic loci. Here, we describe the functions of the main components of the replisome, as well as some of the obstacles to efficient DNA replication that the replisome faces. Taken together, this review summarizes the current understanding of the enormously complicated task of replicating eukaryotic DNA.

Keywords: DNA replication, replisome, replication fork, genome stability, checkpoint, hairpin barriers, hard-to-replicate sites

What is the replication fork?

Our DNA determines everything about us. And for this reason, a copy of our DNA is needed in every cell in our body, except for our red blood cells. A copy of DNA is made just before a cell divides to create two cells. For this DNA replication to take place, the DNA has to be in an orientation that allows the replication machinery to make a copy. Our DNA is double-stranded, and the strands are held together by hydrogen bonds.

The normal structure of our DNA when not copied is a double helix. This looks a lot like a spiral staircase. In this normal form, DNA cannot be copied. DNA helicase is needed to open the DNA and expose the nucleotide bases that are used as a template to replicate the DNA. The area of ​​DNA that the DNA helicase opens is known as the replication fork because it looks a lot like a fork in the road.

The role of the replication fork

The replication fork is the area where DNA replication will actually take place. There are two strands of DNA that are exposed once the double helix opens. One strand is called the leading strand and the other strand is called the lagging strand. The leading strand is exposed in the 5′-3′ direction, while the lagging strand is exposed in the 3′-5′ direction. DNA is always copied in the 5′-3′ direction.

As the main strand is exposed, DNA polymerase will use the main strand as a template to create a continuous complementary strand of DNA. As the lagging strand is exposed, RNA primers are needed to start the replication process. The RNA primer will bind to the most 5′ end of the exposed part of the lagging strand. This primer then allows DNA polymerase to bind and add the complementary strand to the lagging strand in small segments known as Okazaki fragments.

Genetic Variation in Meiosis

Introduction of Genetic Variation in Meiosis

The gametes produced in meiosis are not genetically identical to the initial cell and they are not identical to each other. As an example, consider the meiosis, which shows the end products of meiosis for a simple cell with a diploid number of 2n = 4 chromosomes. The four gametes produced at the end of meiosis II are slightly different, each with a unique combination of the genetic material present in the initial cell.

It turns out that there are many more types of potential gametes than the four, even for a simple cell with only four chromosomes. This diversity of possible gametes reflects two factors: crossing over and the random orientation of meiosis I.

Crossing. The points where homologues cross and exchange genetic material are chosen more or less randomly and will be different in each cell that goes through meiosis. If meiosis occurs many times, as it does in the human ovaries and testes, the crossovers will occur at many different points. This repetition produces a wide variety of recombinant chromosomes, chromosomes in which pieces of DNA have been exchanged between homologues.

Random orientation of homologous pairs. Random orientation of homologous pairs during metaphase of meiosis I is another important source of gamete diversity.

What exactly does random orientation mean here? Well, a homologous pair consists of one homolog from your dad and one from your mom, and you have 23 pairs of homologous chromosomes in total, counting X and Y as homologs for this purpose. During meiosis I, the homologous pairs will separate to form two equal groups, but it is not usually the case that all the paternal (daddy) chromosomes go to one group and all the maternal (maternal) chromosomes go to the other.

Instead, each homologous pair will flip a coin to decide which chromosome belongs to which group. In a cell with only two pairs of homologous chromosomes, like the one on the right, the random orientation of metaphase allows for 22 = 4 different types of possible gametes. In a human cell, the same mechanism allows for 223 = 8,388,608 different types of possible gametes. And that’s not even considering the crossovers!

Given those kinds of numbers, it’s highly unlikely that any two sperm or eggs produced by one person will be the same. It is even more unlikely that you and your sister or brother are genetically identical, unless you are identical twins, thanks to the process of fertilization (in which a single egg cell from mom combines with a single sperm cell from dad, forming a zygote whose genotype is well beyond one in a trillion!).

Meiosis and fertilization create genetic variation by making new combinations of genetic variants (alleles). In some cases, these new combinations can make an organism more or less fit (able to survive and reproduce), thus providing the raw material for natural selection. Genetic variation is important in allowing a population to adapt through natural selection and thus survive in the long term.

Protein Synthesis

The art of protein synthesis

This incredible work of art shows a process that takes place in the cells of all living things: the production of proteins. This process is called protein synthesis and it actually consists of two processes: transcription and translation. In eukaryotic cells, transcription takes place in the nucleus. During transcription, DNA is used as a template to form a molecule of messenger RNA (mRNA). The mRNA molecule then leaves the nucleus and heads for a ribosome in the cytoplasm, where translation occurs. During translation, the genetic code on the mRNA is read and used to make a protein. These two processes are summarized in the central dogma of molecular biology: DNA → RNA → Protein.

Transcription

Transcription is the first part of the central dogma of molecular biology: DNA → RNA. It is the transfer of genetic instructions in DNA to mRNA. During transcription, one strand of mRNA is complemented by one strand of DNA.

Transcription Steps

Transcription takes place in three steps: initiation, elongation, and termination. The steps are illustrated following.

  • Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called a promoter. This tells the DNA to unwind so that the enzyme can “read” the bases on one of the DNA strands. The enzyme is ready to produce an mRNA strand with a complementary base sequence.
  • Elongation is the addition of nucleotides to the mRNA chain.
  • Termination is the end of the transcript. The mRNA chain is complete and separates from the DNA.

mRNA processing

In eukaryotes, the new mRNA is not yet ready for translation. At this stage, it is called pre-mRNA and must go through more processing before it leaves the nucleus as mature mRNA. Processing may include splicing, editing, and polyadenylation. These processes modify mRNA in several ways. Such modifications allow a single gene to be used to produce more than one protein.

  • Splicing removes the introns from the mRNA. Introns are regions that do not code for protein. The remaining mRNA consists only of regions called exons that code for protein. The ribonucleoproteins in the diagram are small proteins in the nucleus that contain RNA and are necessary for the splicing process.
  • The editing changes some of the nucleotides in the mRNA. For example, a human protein called APOB, which helps transport lipids in the blood, has two different shapes due to editing. One form is smaller than the other because the editing adds an earlier stop signal on the mRNA.
  • Polyadenylation adds a “tail” to the mRNA. The tail consists of a chain of As (adenine bases). It marks the end of the mRNA. It is also involved in the export of mRNA from the nucleus and protects the mRNA from enzymes that might break it down.

Translation

The translation is the second part of the central dogma of molecular biology: RNA → Protein. It is the process in which the genetic code in mRNA is read to make a protein. After the mRNA leaves the nucleus, it moves to a ribosome, which consists of rRNA and proteins. The ribosome reads the sequence of codons on the mRNA, and the tRNA molecules bring the amino acids to the ribosome in the correct sequence.

To understand the role of tRNA, you need to know more about its structure. Each tRNA molecule has an anticodon for the amino acid it carries. An anticodon is complementary to an amino acid codon. For example, the amino acid lysine has the codon AAG, so the anticodon is UUC. Therefore, the lysine would be carried by a tRNA molecule with the anticodon UUC. Whenever the AAG codon appears on the mRNA, a tRNA UUC anticodon is temporarily attached. While binding to the mRNA, the tRNA gives up its amino acid. With the help of rRNA, bonds are formed between amino acids as they are carried one by one to the ribosome, creating a polypeptide chain. The amino acid chain continues to grow until a stop codon is reached.