Identification of β Clamp-DNA Interaction Regions That Impair the Ability of E. coli to Tolerate Specific Classes of DNA Damage

Abstract

The E. coli dnaN-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.

The SOS system: A complex and tightly regulated response to DNA damage

“…Therefore, the β‐clamp plays a pivotal role during the TLS process acting as the common platform to which the TLS polymerases are directed when normal replication is stalled at a lesion site. Discrete β clamp‐DNA interaction regions were shown to contribute to its ability to manage actions of its different partner proteins, particularly Pol IV and Pol V, allowing E. coli to tolerate specific classes of DNA damage (Nanfara et al, ). It has been reported by Indiani et al () that RecA protein acts as a switch to regulate the occupancy of polymerases in a moving replication fork during TLS.…”

Escherichia coli DNA replication: the old model organism still holds many surprises

“…A possibility has been raised that these alternative binding sites might facilitate the rapid exchange of DNA polymerases during replication. Interestingly, β 2 mutants carrying mutations within the DNA-binding region have been isolated and shown to somehow affect interactions with Pol III or Pol II and Pol IV (Heltzel et al 2009 , Homiski et al 2021 , Berger and Cisneros 2023 ) and even impair the ability of E. coli to tolerate DNA damage (Nanfara et al 2016 ).…”

Distal Mutations in the β-Clamp of DNA Polymerase III* Disrupt DNA Orientation and Affect Exonuclease Activity

“…The binding interface between the two monomers consists of predominantly hydrophobic residues. Many mutagenesis studies have been carried out to understand the importance of the β-clamp and its role in DNA replication. − …”