Understanding Molecular Biology Part II – DNA Replication (If you missed Part I, check it out before going on to Part II below.)

As a cell grows and gets ready to divide in two, its DNA must duplicate so that each new daughter cell receives a complete genome. To do this, the double-stranded DNA of each chromosome separates, and every single strand is copied to regenerate the double-stranded forms. This copying process requires many cellular proteins that aggregate to form a replication complex on the DNA at certain locations called origins of replication (Fig.1). There are too many proteins involved to discuss them all, so I’ll only mention three of the important players in this process. The first is the origin recognition complex (ORC) which consists of a group of six different proteins bound together. ORC recognizes sequence and structural features of the DNA present at the origins of replication and binds to the origin to start the replication process. The binding of ORC to the origin allows additional proteins to assemble at the location and eventually form the functional replication complex. Another important component of the replication complex is known as the MCM complex, also an assemblage of six different proteins. MCM is an enzyme known as a helicase. Helicases are enzymes that can use energy to separate the double-stranded DNA at the origin to create a single-stranded “bubble”. This bubble has two single strands available for copying. If you think of the double-stranded DNA as a closed zipper, the MCM complex is like the slider that moves along and separates the strands. MCM complexes form at both ends of the bubble and act to separate the DNA in both directions. Lastly, the actual synthesis of the new strands is done by enzymes known as DNA polymerases. These enzymes use the single strands as templates and join together individual deoxyribonucleotides to form the new complementary strands.

Fig. 1: Initiation of DNA Replication. Chromosomes have multiple regions on their DNA that serve as the locations where new DNA synthesis begins. These regions are defined by unique sequences and structural features. Shown in A. is a short stretch of DNA with the origin region indicated. . The origins of replication are recognized by the Origin Recognition Complex (ORC) which consists of a group of 6 different proteins that assemble and bind to the origin region (B.). The binding of ORC recruits additional proteins that collectively form the replication complex. One of the recruited components is MCM, again a complex of 6 different proteins. MCM has helicase activity which allows it to pull apart the double-stranded DNA to form the single-stranded replication bubble (C.). MCM complexes assemble at both ends of the bubble and separate the DNA in opposite directions. DNA polymerase (DNAP) is the enzyme that synthesizes the new DNA using each single strand as the template. Created with BioRender.com.

Human chromosomes are large so they contain multiple origins of replication. Eventually, the replication complexes formed at these sites copy all of the original double-stranded DNA to yield two double-stranded DNAs. Each of the two final double-stranded DNAs contains one strand from the original DNA and one newly synthesized strand. This process happens on all 23 of our chromosomes so at completion the cell has duplicated the entire genome and is ready to divide. As the original cell splits into two daughter cells, one copy of the complete genome goes to each daughter cell (Fig. 2). Each daughter cell can then grow and eventually divide itself as needed.

Fig. 2: Cell Division. Shown in A. is a cell with two chromosomes in its nucleus. Each chromosome is a pair of paternal and maternal copies joined at the centromere. As the cell grows the DNA will replicate and the chromosomes are duplicated to yield two complete copies (B.). As the cell undergoes division (C.), the duplicated chromosomes separate so that each daughter cell will have one complete genome. Created with BioRender.com.

During this replication process, sometimes the DNA polymerase makes mistakes and puts down the wrong deoxyribonucleotide, for example, pairing a G with a T rather than putting in an A that should pair with T (Fig. 3). Exposure to radiation and certain chemicals can also damage the DNA and cause sequence errors. Our cells have multiple DNA repair systems that try to correct these errors, but nothing is perfect and so permanent errors in our DNA do occur. Such errors are mutations, and they accumulate in our genomes throughout our lives as our cells divide and as we are exposed to DNA-damaging agents in our environment. Mutations in certain genes can cause cancers, so our cancer risk increases as we age because more and more of our cells have mutations. Since these mutations are random, any cell type could be affected, and cancer can occur in any of our organs. These random mutations can also damage any of our genes which contributes to the slow decay of our bodily functions, i.e. aging.  Ultimately, the very process of DNA replication which is essential to the growth and replenishment of our bodies also contributes to our eventual demise.

Fig. 3: Replication Error. Shown in A. is the initial double-stranded DNA with the upper (U) and lower (L) strands designated. During replication, the double-stranded DNA will be separated by the MCM complex to form the single-stranded templates (B.). As replication proceeds, the upper strand is correctly copied to yield a new double-stranded DNA (C.) composed of the original upper strand and a new lower strand (NL). As the original lower strand is copied (D.) a replication error occurs that pairs a T nucleotide on the lower strand with a G nucleotide on the new upper strand (NU) rather than the correct A nucleotide. This change in the nucleotide sequence on the new upper strand is a mutation. Created with BioRender.com.

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